Blood-brain barrier targeting antibodies

ABSTRACT

This invention provides antibodies that bind brain endothelial cell receptors resulting in endocytosis/transcytosis of the receptor and bound ligands. In some embodiments, the ligand comprises the antibody in combination with a pharmaceutically active compound and the antibody directs delivery of the compound across the blood brain barrier (BBB). The invention also provides methods of identifying endothelial cell specific antibodies by panning the library against cultured cell monolayers. The invention further allows for identifying endothelial cell receptors that bind the antibody thereby providing target receptors against which to isolate further cognate ligands and their associated transport systems and by which to identify transcytosis transporters targeted by the antibodies.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/540,897, filed on Aug. 3, 2009, which is a continuation-in-part ofU.S. application Ser. No. 11/759,723, filed Jun. 7, 2007, which claimsthe benefit of U.S. Provisional application 60/811,618, filed Jun. 7,2006. All three of these applications are incorporated herein byreference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under NS052649 andEY018506 awarded by the National Institutes of Health, and under 0238864awarded by the National Science Foundation. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to antibodies recognizing endothelialcell surface receptors and methods of identifying such antibodies.

BACKGROUND OF THE INVENTION

Treatment modalities for brain and neurological diseases are extremelylimited due to the impermeability of the brain's blood vessels to mostsubstances carried in the blood stream. The blood vessels of the brain,referred to collectively as the blood-brain barrier (BBB), are uniquewhen compared to the blood vessels found in the periphery of the body.Tight apposition of BBB endothelial cells (EC) to neural cells likeastrocytes, pericytes and neurons induces phenotypic features thatcontribute to the observed impermeability. Tight junctions between ECscomprising the BBB limit paracellular transport, while the lack ofpinocytotic vesicles and fenestrae limit non-specific transcellulartransport. These factors combine to restrict molecular flux from theblood to the brain to those molecules that are less than 500 daltons andalso lipophilic. Thus, using the large mass transfer surface area (over21 m² from 400 miles of capillaries in human brain) of the bloodstreamas a delivery vehicle is largely infeasible except in thosecircumstances where a drug with the desired pharmacological propertiesfortuitously possesses the size and lipophilicity attributes allowing itto pass freely through the blood vessel. Because of such restrictions,it has been estimated that greater than 98% of all small moleculepharmaceuticals and nearly 100% of the emerging class of protein andgene therapeutics do not cross the BBB.

In addition to the physical barrier presented by the BBB, effluxtransporters such as p-glycoprotein (MDR1) and members of the multi-drugresistance-associated protein family (MRP) serve to further limit brainuptake of even those small molecules that are small and lipophilic. FIG.1A is a micrograph of a section of rat brain (V=ventricle) illustratingthe sequestration of horseradish peroxidase in vessels, while in smallbrain regions perfused by capillaries lacking the BBB, the proteindiffuses readily into brain tissue. FIG. 2A also illustrates theimpermeable nature of the BBB: histamine (111 Da) remains sequesteredwithin the blood vessels and does not enter the brain interior.

Although neurological diseases such as brain cancer, Alzheimer'sdisease, Parkinson's disease, and stroke continue to afflict peopleworldwide, there has been a paucity of new therapies to treat suchdiseases. Lack of treatment modalities can, in part, be attributed tothe lack of effective brain delivery strategies. Due to this lack oftreatment methods the National Institutes of Health tumor and strokeprogress review groups have even identified the search for innovativestrategies for drug or gene targeting through the blood-brain barrier asa top research priority. The National Institutes of Health (NIH)guidelines instruct that such breakthroughs in basic neuroscience can bedelivered to the clinic and “require an agent delivery strategy and/orthe ability to target specific areas of the brain”. Thus, in the absenceof appropriate vehicles for targeting and trans-BBB transport, thepipeline of new CNS medicines will likely continue to be inadequate forthe people suffering from neurological disorders. As examples, proteintherapeutics known as neurotrophins have been investigated recently fortheir protective capacity in stroke, reversal of Parkinson's diseasesymptoms after direct infusion into the brains of human subjects, andfor their ability to direct specialized differentiation of neural stemcells for potential treatment of Parkinson's disease and otherneurodegenerative diseases such as Alzheimer's disease, Huntington'sdisease and multiple Sclerosis. Although extremely promisingtherapeutics, these trophic factors do not readily cross the BBB andwill require a noninvasive delivery system for widespread, effectiveadministration.

Present brain delivery strategies are particularly invasive and requirecircumvention of the BBB. Strategies requiring neurosurgery are used forimplantation of polymer particles infused with drug, but the treatmentvolume is limited because the extracellular fluid in brain tissue isquiescent, and simple molecular diffusion only allows for a modestpenetration distance of 2-3 mm. A similar method is direct injection ofa drug into the brain ventricles, but the penetration into brain tissueis also limited for such intraventricular injections because thecerebral spinal fluid is rapidly cleared and is turned over 4-5 timesdaily. In addition, protein therapeutics, such as, for example,Glial-cell Line-Derived, Neurotrophic Factor (GDNF) for Parkinson'sdisease have been delivered through neurosurgically implanted catheterswith continual drug infusion by a peripheral pump. Disruption of the BBBhas been investigated using hyperosmolar solutions and vasoactive agentslike serotonin and bradykinin peptides to allow free passage ofmolecules from the blood to the brain. This method is primarily usedclinically only in terminal patients because alterations in the BBB canlead to toxic effects due to the free access of solutes and immunefactors that are normally excluded from brain.

The aforementioned invasive strategies can have success for thosediseases with limited treatment volumes, such as, for localized,non-metastatic brain tumors. However, for chronic conditions requiring arepetitive treatment regimen, or for those diseases present in largeportions of the brain such as Alzheimer's disease, a noninvasive drugdelivery strategy would be substantially more preferable and practical.

A variety of noninvasive brain drug delivery methods have beeninvestigated that make use of the brain blood vessel network to gainwidespread drug distribution. The brain capillary network has an averagespacing of just 40 microns between capillaries and is sufficiently densethat each brain cell essentially has its own vessel for nutrient supply(FIG. 2). In addition, if the endothelial barrier of the BBB can beovercome such that a drug is deposited on the brain side of the BBB, thediffusion distance is short enough that each brain cell should beaccessible to the drug. As a result, in contrast to invasive methods, acomprehensive treatment volume can result. These noninvasive transportsystems/mechanisms can be generally clustered into three groups: 1,non-specific uptake; 2, carrier-mediated transport; and 3,receptor-mediated transport (FIG. 3).

Non-specific uptake mechanisms, while allowing some transport across theBBB, lack sensitivity and specificity. Cationic protein transductiondomains fall into the realm of non-specific carriers, and although theHIV TAT peptide was shown to gain access to the brain interstitium afterintraperitoneal injection, subsequent pharmacokinetic analysis indicatedthat the rapid clearance and broad organ uptake would necessitate veryhigh doses to gain a pharmacologic effect. Another non-specific uptakemechanism is the surfactant coating of nanoparticles with polysorbate80. Although the mechanism of brain uptake is still unresolved, thelabile nature of the particles in vivo leads to short-livedpharmacologic effects and possible BBB permeabilization. Both of thesenon-specific methods suffer from a lack of selective targeting andresult in widespread distribution of the active compound throughout thebody with concomitant systemic effects.

Carrier-mediated drug transport relies on the presence of endogenoustransmembrane proteins that are selective and stereospecific for smallmolecule solutes. For instance, L-dopa, administered to treatParkinson's disease gains entry to the brain by utilizing the largeamino acid transporter (LAT-1). The successful transport through theblood-brain barrier is a result of L-dopa mimicking the structure ofphenylalanine with only the substitution of two hydroxyl groups on thearomatic ring of phenylalanine Utilization of the saturable biotintransport system for delivery of biotinylated drugs has also beenattempted. In addition, it is likely that the efflux of the AIDS drugAZT progresses in a carrier-mediated fashion. However, due to thestereospecificity and steric constraints imposed by these selectivemembrane pores, applications are potentially limited.

Receptor-mediated transport involves the binding of an exofacial epitopeof a cell surface receptor and triggering of an energy intensivetranscellular transport process known as transcytosis (FIG. 3). Drugscan be delivered using these portals if conjugated to the natural ligandor an antibody that can trigger the transcytosis process. This methodhas been successful in allowing for non-invasive transport of smallmolecules, proteins, genes, nanoparticles, and liposomes up to 100 nm insize. The receptors that are commonly targeted for transcytosis are thelow density lipoprotein (LDL) receptor, the transferrin receptor, andthe insulin receptor. Similar less specific processes involvingabsorptive-mediated transcytosis have been used with cationized proteinsthat promote receptor clustering and activation of the transcytosispathway. Strategies oftentimes target the cell surface receptor in waysthat do not disrupt the normal transport of endogenous ligand. Thereforethe impact on normal metabolic pathways is limited. In addition, sincetranscytosis employs the vesicular trafficking system, this strategy isnot nearly as limited by the size and shape constraints ofcarrier-mediated transport.

Antibodies are particularly well suited for targeting BBBreceptor-mediated transcytosis systems given their high affinity andspecificity for their ligands. As examples, appropriately-targetedantibodies that recognize extracellular epitopes of the insulin andtransferrin receptors can act as artificial transporter substrates thatare effectively transported across the BBB and deposited into the braininterstitium via the transendothelial route. Additionally, whenconjugated to drugs or drug carriers of various size and composition,the BBB targeting antibodies mediate brain uptake of the therapeuticcargo. Noninvasive transport of small molecules such as methotrexate hasbeen achieved using anti-transferrin receptor antibodies. Proteins suchas nerve growth factor, brain derived neurotrophic factor, and basicfibroblast growth factor were delivered to the brain after intravenousadministration by using an anti-transferrin receptor antibody. Thelatter two cases promoted reduction in stroke volume in rat middlecerebral artery occlusion models. Liposomes and liposomes containinggenes have also been delivered to the brain in vivo usinganti-transferrin receptor antibodies. In particular, gene-containingantibody-targeted liposomes have been targeted to rat brain forrestoration of tyrosine hydroxylase activity in an experimentalParkinson's disease model and to primate brain using a humanizedanti-insulin receptor antibody. In addition, brain delivery of the newclass of RNA interference drugs via pegylated immunoliposomes has beendemonstrated to increase survival of mice implanted with an experimentalhuman brain tumor model. Further, anti-transferrin receptor conjugatednanoparticles have been produced. Finally, even if an antibody binds tothe brain microvasculature or internalizes without full transcytosis, itcan have drug delivery benefits. When conjugated to a liposome ornanoparticle loaded with lipophilic small molecule drugs, it might bepossible to raise the local BBB concentration of drug and helpcircumvent brain efflux systems, thereby facilitating brain uptake.Finally, in the event binding occurs without transcytosis or eveninternalization, the identification of BBB-specific antibody receptorsor ligands will further help to characterize and identify components ofthe transporter system and help to further optimize antibodies that dointernalize and trancytose. Taken together, these results indicate thepotential utility of antibody-targeted transcytosis systems fornoninvasive trafficking of drugs into the brain.

Regardless of the promise shown for antibody mediated transport in priorstudies, the current antibody targeting reagents lack specificity andtransport efficiency. Although early results derived from thereceptor-mediated transcytosis process are promising due to itsrobustness in delivery of drugs or drug carriers in many formats, italso has some serious drawbacks that need to be addressed for generalclinical success. The present methods rely on receptors that areubiquitously expressed like the transferrin and insulin receptors. Thisleads to mis-targeting of potentially expensive drugs that also may haveunwanted side effects in tissues other than the brain. In addition, thepresent methodologies generally result in a low fraction (1-4%) of theinjected dose actually reaching the brain target as a consequence ofpoor targeting and nonideal BBB permeability. This loss of between96-99% of the administered therapeutic could hamper the development ofthese delivery approaches given the cost of drug manufacture, especiallyfor protein and gene-based medicines that currently comprise nearly 700drugs in various stages of clinical trials. Finally, the antibodies usedin the aforementioned proof-of-concept experiments are either of murineorigin or partially humanized, and this could lead to unwantedimmunogenic reactions in human patients. Therefore, the identificationof fully human antibodies that specifically recognize brain endothelialreceptors would vastly improve the targeting and efficiency of drugdelivery while minimizing side-effects.

SUMMARY OF THE INVENTION

This invention provides antibodies that bind endothelial cell receptorsresulting in endocytosis of the receptor and bound ligands. In oneexemplary embodiment, the invention comprises an isolated antibodyfragment having the amino acid sequence set forth in any one of SEQ IDNOs:1-34. In another embodiment, the invention provides an isolatednucleic acid having a sequence coding for an amino acid as set forth inany one of SEQ ID NOs:1-34. In some preferred versions, the isolatedantibody fragment is a single chain fragment variable (scFv) fragment.In other preferred versions, the isolated antibody is a Fab, an IgG orany other ligand specific to the endothelial cell receptor.

In another preferred embodiment, the invention is a pharmaceuticalcomposition comprising an antibody linked to a pharmaceutically activecompound that is useful in transferring the pharmaceutically activecompound across the blood brain barrier (BBB).

In another exemplary embodiment, the invention is an isolated expressionvector that includes a polynucleotide encoding the amino acid sequenceset forth in SEQ ID NOs:1-34. In other embodiments, the inventionincludes a purified and isolated host cell comprising the expressionvector containing the isolated nucleic acid encoding the amino acidsequence set forth in any one of SEQ ID NOs:1-34. It should beappreciated that the host cell can be any cell capable of expressingantibodies, such as, for example fungi, mammalian cells, including theChinese hamster ovary cells; insect cells, using, for example, abaculovirus expression system; plant cells, such as, for example, corn,rice, Arabidopsis, and the like.

In yet another exemplary embodiment, the invention comprises a processfor expressing an antibody fragment capable of binding to a brainendothelial cell receptor comprising: (a) displaying an antibodyfragment on a yeast cell; (b) panning the antibody displaying yeast cellagainst a brain endothelial cell culture; (c) identifying displayedantibody fragments that specifically bind brain endothelial cellreceptors; (d) inserting an isolated nucleic acid coding for theantibody fragment identified in step (c) in an expression vector; and(e) transforming a host cell with the expression vector. In someversions of this embodiment, the host cell is selected from the groupconsisting of: yeast, bacteria, and combinations thereof. In somepreferred embodiments the host cell is Saccharomyces cerevisiae or E.coli.

In another exemplary embodiment, the invention is a method ofidentifying a brain endothelial cell specific antibody. This embodimentincludes displaying antibody fragments on a yeast cell surface, panningthe displayed antibody against a brain endothelial cell culture,isolating specific binders to the membrane receptors on the brainendothelial cell, and identifying the specific binders, therebyidentifying an endothelial cell specific antibody. In some preferredembodiments the endothelial cell culture is provided in a cellmonolayer.

In still another exemplary embodiment, the invention comprises a methodof identifying endothelial cell receptors functioning in or related toendocytosis and transcytosis comprising, providing a culturedendothelial cell monolayer and panning a yeast-displayed antibodylibrary against the endothelial cell monolayer, isolating antibody boundendothelial cells; and identifying the cognate receptor.

Novel human antibodies that target BBB transport systems have immenseutility. Unlike the antibody delivery strategies that utilized wellstudied systems such as the transferrin and insulin systems, identifyingnovel receptor-mediated transport systems is quite difficult given thatthese ligands are membrane proteins that have yet to be identified.Therefore, the inventors have developed a novel selection methodology toidentify fully human antibodies having the required functionality of BBBbinding. The power of this technique is the concomitant selection ofpotential drug delivery vector and its cognate cell surface receptor(receptor-mediated carrier system) with no prior knowledge as to theorigin of either component. FIG. 4 depicts a general scheme for theselection of such antibodies according to the invention.

Another aspect of the present invention is in regard to antibody targetsat the blood brain barrier, particularly, targets that represent themolecular machinery for internalization (endocytosis) and transcytosis.Accordingly, the present invention is directed to a purified scFvAantigen having a molecular weight of approximately 124 kDa whenimmunoprecipitated by human scFvA under non-reducing conditions. Theinventors identified and demonstrated this antigen is expressed inintact brain capillaries.

Accordingly, the invention further encompasses an antibody-blood brainbarrier transporter system. Such a system includes: (a) an antigenhaving a molecular weight of approximately 124 kDa whenimmunoprecipitated by human scFvA under non-reducing conditions, theantigen expressed in intact brain capillaries; and (b) a purifiedantibody bound to the scFvA antigen. The purified antibody has the aminoacid sequence set forth in any one of SEQ ID NOs:1-5, more preferablythe antibody is scFvA having the amino acid sequence set forth in SEQ IDNO:1. In certain embodiments, the purified antibody is provided incombination with a pharmaceutically active compound.

As demonstration of a similar exemplary embodiment, the inventors haveidentified the scFvJ antibody's (SEQ ID NO:14) target antigen as neuralcell adhesion molecule (NCAM). Thus, the invention further encompassesan antibody-blood brain barrier transporter system including (a) anantigen comprising NCAM; and (b) a purified antibody bound to theantigen. In certain embodiments, the purified antibody has the aminoacid sequence set forth in SEQ ID NO:14. Preferably, the purifiedantibody is provided in combination with a pharmaceutically activecompound.

Based upon the disclosed antibody-blood brain barrier transportersystem, the invention also contemplates a method of delivering apharmaceutically active compound across the blood brain barrier to asubject's brain. Such a method includes administering a pharmaceuticallyactive compound in combination with a purified antibody having an aminoacid sequence of any one of SEQ ID NOs:1-5, SEQ ID NOs:11-12, SEQ IDNO:15, and SEQ ID NO:21 to a subject such that the antibody directsdelivery of the pharmaceutically active compound across the blood brainbarrier to the subject's brain.

These and other features and advantages of various exemplary embodimentsof the articles and methods according to this invention are describedin, or are apparent from, the following detailed description of variousexemplary embodiments of the methods according to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary embodiments of the methods of this invention will bedescribed in detail, with reference to the following figures, wherein:

FIGS. 1A and 1B are micrographs illustrating the impermeability of theblood-brain barrier. FIG. 1A shows that horseradish peroxidase issequestered in brain blood vessels and does not access the parenchyma,except for in the small circumventricular organs that lack a BBB (medianeminence above). FIG. 1B. illustrates the effectiveness of the BBB:radio-labeled histamine, only 111 Da, cannot enter the brain or spinalcord of the mouse.

FIG. 2 is an electron micrograph of a human cerebellar cortex vascularcast illustrating the extent of the vascular network. Scale bar is 40μm.

FIG. 3 is a schematic representation of various BBB transport options.

FIG. 4 is a flowchart of one embodiment of antibody-mediated brainendothelial cell transcytosis.

FIG. 5 is a cartoon of an immunoglobulin molecule illustrating theposition of the variable regions and the origin of the single-chainantibody variable fragment (scFv). Variable light and heavy regions areconnected by a flexible polypeptide linker and comprise the minimalbinding subunit of an intact antibody.

FIGS. 6A-D are schematic representations of yeast surface display of anscFv construct (4-4-20) whose antigen is fluorescein. The scFv proteinis fused to the self-assembling, mating agglutinin proteins (Aga1p andAga2p) allowing surface display of scFv proteins that actively bindfluorescein on the surface of the yeast particle. FIG. 6A is a phasecontrast image of yeast displaying 4-4-20. FIG. 6B is a fluorescenceimage of yeast displaying 4-4-20 bound to fluorescein-dextran(FITC-dextran). FIG. 6C is a phase contrast image of yeast displayingirrelevant scFv, D1.3. FIG. 6D is a fluorescence image of D1.3 yeastthat fails to bind FITC-dextran.

FIG. 7 is a phase contrast image showing the specific interactionbetween yeast displaying 4-4-20 scFv and FITC-labeled RBE4 cells.Conjugation of 4-4-20 yeast cells with biotinylated and neutravidin-FITC(NAFITC) labeled (top row) or unlabeled (bottom row) RBE4 cells. Yeastand RBE4 cells can be distinguished by their size, 4 μm and 20 μmrespectively. Images of the same microscope fields were taken before andafter each washing step. Scale bar: 50 μm. As shown, the FITC labeledcells exhibit continued binding to yeast after 3 washes while thenon-FITC labeled cells do not.

FIG. 8 is a bar graph showing the effects of scFv affinity on multipleround enrichment. Mixtures containing 4-4-20, 4M5.3 and D1.3 yeast(1:1:1×10⁵) were panned against NAFITC-labeled RBE4 cell monolayers.After each round, the fractions of 4-4-20 and 4M5.3 in the mixture wereevaluated by flow cytometry. Solid bars correspond to 4-4-20 percentageand hatched bars correspond to 4M5.3 percentage. The dotted lineindicates the detection limit of the flow cytometry assay.

FIG. 9 is a schematic representation illustrating the overall strategyof biopanning to identify BBB-binding and internalizing scFv.

FIG. 10 illustrates the enrichment of RBE4-binding scFv's via biopanning

FIG. 11 is a schematic diagram of the cell monolayer panning and highthroughput yeast clone profiling strategy. 11A, yeast growth in 96-wellplate; 11B, yeast are added to RBE4 culture; 11C, induced yeast; 11D,non-induced; 11E initial PCR or binders; 11F Restriction digestproviding “fingerprint” of binders and identifying germline family; 11Gsequencing of unique scFv clones.

FIG. 12 is a table summarizing the binding of scFv variants. (a) UniquescFv were clustered by homology. Each class of scFv antibodies differsfrom all other classes by at minimum one CDR3 (20% amino acid homology).The CDR3 regions were focused upon for defining classes given thenonimmune basis for the library that limits CDR1 and CDR2 diversity andthe fact that CDR3 of the VH and VL play a major role in determiningbinding specificity and affinity. Within a class, CDR1, CDR2, and CDR3(>85% amino acid homology) of VH and VL all have high homology. (b) AnscFv was deemed unique by amino acid sequence and BstN1 digest pattern.(c) The germline antibody gene usage was determined by subjecting thededuced scFv amino acid sequence to IgBLAST(http://www.ncbi.nlm.hig.gov/igblast/). (d) Clones subtracted in theVHCDR2 yeast Northern blot experiments were counted in the totals ofeither scFvA or scFvD based on their frequency of occurrence in smallscale screens, and therefore represent estimates.

FIG. 13 is a sequence alignment of scFv clones B, G, C and K that showsequence homology to clone scFv A. VH—variable heavy region; VL—variablelight region; CDR—complimentarity determining region.

FIG. 14 is a sequence alignment of scFv clones D, I and H that sharesequence homology. VH—variable heavy region; VL—variable light region;CDR—complimentarity determining region.

FIG. 15 is a sequence alignment of scFv clones A, C, B, G, K and Fillustrating that F has a similar homology to the variable light (VL)domains but not to the variable heavy (VH) domain. VH—variable heavyregion; VL—variable light region; CDR—complimentarity determiningregion.

FIG. 16 is a polyacrylamide gel (top panel) showing that the secretedproteins have the appropriate weight for an scFv and a table (bottompanel) summarizing the binding and internalization of scFv by RBE4cells.

FIG. 17 is a collection of micrographs illustrating dual fluorescentstaining of RBE4 binding scFv's showing specific binding andinternalization of scFv A by RBE4 cells but not scFv D.

FIG. 18 provides equilibrium binding attributes of scFvA and scFvD. Leftpanel: binding isotherm for scFvA interaction with live RBE4 cells. Theplot shows the fitted monomeric equilibrium binding functions andexperimental data from two independent experiments. Right panel: bindingisotherm for dimerized scFvD interaction with RBE4 cells. The plot showsthe fitted monomeric equilibrium binding functions used to generate anapparent affinity (avidity) and experimental data from two independentexperiments. Insets indicate raw flow cytometry histograms that wereused to generate the binding curves.

FIG. 19 depicts yeast display immunoprecipitation of antigens for scFvA(A), scFvD (D), scFvJ (J) and OX26 scFv (O). Irrelevant anti-hen egglysozyme scFv (N) was used as a negative control. Theimmunoprecipitation products were resolved by either nonreducing (NR) orreducing (R) gel electrophoresis and were probed with an anti-biotinantibody or an anti-transferrin receptor antibody (OX26).

FIG. 20 are fluorescence micrographs allowing evaluation of scFv bindingand internalization properties. RBE4 cells were labeled with purified,pre-dimerized scFvA, scFvD, irrelevant scFv 4-4-20 or OX26 monoclonalantibody at 4° C. for cell surface labeling and then shifted to 37° C.to promote cellular trafficking. The cells were then labeled withAlexaFluor 555 conjugated anti-mouse IgG at 4° C. followed by AlexaFluor488 conjugated anti-mouse IgG with or without cell permeabilization bysaponin (SAP) treatment. Merged images of the AlexaFluor-labeled imagesare shown. Scale bar: 20 μm.

FIG. 21 are fluorescence micrographs showing that scFv A recognizes abrain endothelial antigen expressed in vivo. Frozen rat brain sectionswere co-labeled with scFvA (1) or 4-4-20 (4) and the brain endothelialcell marker GSA-FITC (2, 5). (3, 6) are merged images indicating theoverlap between scFv and GSA-FITC labeling. In contrast to scFvA,irrelevant scFv 4-4-20 did not yield any labeling. Similar results wereobserved for mouse brain sections and freshly isolated capillaries.Finally, much like the ranking of antigen density for RBE4 cultures,qualitative labeling intensities indicated that the scFvA antigendensity in vivo was higher than that for the transferrin receptor (datanot shown). Scale bar: 50 μm.

FIG. 22 shows schematics of the relevant steps of the YDIP procedure. Instep (1), target cells containing the antigen are lysed usingdetergents. Cell surface antigens such as membrane proteins aresolubilized by the detergent. Yeast cells that have been induced forsurface display of scFvs in step (2) are combined with the cell lysatefrom (1). In step (3), components in the cell lyste that are not boundto scFvs are washed away, and the yeast cells are collected bycentrifugation. At this point, antibody-antigen characteristics can bequantified by flow cytometry (step 4). In step (5), denaturingconditions such as lowering the pH are used to dissociate theantigen-antibody interaction. The collected antigen can then be analyzedfor antigen identification and characterization in step (6).

FIG. 23 shows ScFv binding activity in detergent solutions. (A)Equilibrium binding titration curves of yeast-displayed D1.3 scFv withHEL in PBS and containing the indicated detergents. The lines representfitted solutions using least-squares regression with a standardbimolecular equilibrium binding model. BSA fixed indicates binding datausing formaldehyde-fixed yeast. (B) Comparison of absolute levels ofbiotinylated HEL binding in various detergent solutions using unfixed(open bars) or formaldehyde-fixed (grey bars) yeast. HEL binding to D1.3on the surface of yeast was detected by using a fluorescentstreptavidin-phycoerythrin conjugate and quantitated by flow cytometryas the mean fluorescence (HEL binding) per cell. Data were normalized tothe case of PBS-BSA. (C) Detection of antigen-antibody interaction forscFvA, scFvK, scFvD and scFvJ. Yeast cells displaying each scFv werelabeled with biotinylated RBE4 cell lysate generated by 1% TX. Yeastdisplaying an irrelevant antibody were used as a negative control (NC).The inset shows an anti-biotin Western blot of antigens isolated usingYDIP from biotinylated RBE4 cell lysate generated using OG. Lane 1:irrelevant scFv, lane 2: scFvA, lane 3: scFvK, lane 4: scFvD, and lane5: scFvJ. (D) Measurement of relative affinities of scFvk by titratingRBE4 cell lysate generated with 1% OG. X-axis indicates theconcentration of RBE4 lysate in terms of percent of a cell lysatecontaining approximately 2 mg of total protein. The light-chain CDR3amino acid sequence of scFvA and scFvK and the relative affinity (K_(d))calculated as described above are indicated in the inset. Error barsrepresent standard deviations from two (A and D) or three (B and C)independent measurements.

FIG. 24 shows recovered antigen purity and quantity by YDIP. (A)Comparison of elution methods from formaldehyde-fixed yeast cellsdisplaying D1.3 scFv. The amount of biotinylated HEL recovered aftereach elution was quantified by Western blot. Elutions were performedusing 3 M NaCl (High Salt), 0.2 M glycine-HCl pH 2.4 (Low pH), 0.2 MNaOH (High pH), 9 M urea (Urea), or 0.2% SDS in 0.1 M Tris (SDS).Elution with PBS-BSA was used as a negative control. The datarepresented mean±S.D. from triplicate measurements expressed as thepercentage of maximum theoretical immunoprecipitation yield. (B) Yeastimmunoprecipitation of HEL from RBE4 cell lysate with low pH elution.HEL was added to RBE4 cell lysates at a concentration of 34 nM and YDIPwas performed. Immunoprecipitation products were separated usingSDS-PAGE and subsequently silver-stained. YDIP was performed usingeither D1.3-displaying yeast (lanes 2 and 4) or an irrelevant 4420 scFvdisplaying yeast (lanes 1 and 3). Lanes 1 and 2 are YDIP products fromunfixed yeast, and lanes 3 and 4 are from formaldehyde-fixed yeast. Lane5 contains 25 ng of purified HEL for comparison. The gel was quantifiedby densitometric analysis.

FIG. 25 shows serial elution method of antigen concentration. (A) ThescFvJ antigen was immunoprecipitated from biotinylated RBE4 cell lysateusing fixed yeast. Elution was performed by adding 50 μL of 0.2 Mglycine-HCl buffer (pH 2.0) to serially elute batches of 6×10⁷immunoprecipitated yeast. Each lane number corresponds to the number ofyeast batches eluted. (B) Densitometric quantitation of (A).

FIG. 26 shows VDIP coupled with MS/MS for antigen identification. (A)Silver-stained gel showing the scFvJ YDIP product separated by SDS-PAGE.Lane 1: raw cell lysate, Lane 2: YDIP using yeast displaying irrelevantscFv, Lane 3: YDIP using yeast displaying scFvJ. Althoughcoomassie-stained gels were used for MS/MS experiments, a silver-stainedgel is shown for clarity. The scFvJ antigen band that corresponds to theband identified by anti-biotin Western blotting is indicated by anarrowhead. (B) MS/MS results showing the two peptides sequenced thatcorrespond to NCAM. All the y-ions identified are indicated on thefigure along with the m/z values. The Mascot score of each ion is alsoindicated and as defined in Table 2, the NCAM identification has aconfidence level of p<0.05. (C) Confirmation of antigen identificationby Western blotting. The left panel (lanes 1-3) was probed with ananti-biotin antibody and the right panel (lanes 4-6) was probed with ananti-NCAM antibody. Lanes 1 and 4: raw biotinylated RBE4 cell lysate,lanes 2 and 5: VDIP product from irrelevant scFv, lanes 3 and 6: VDIPproduct from scFvJ.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is based on the observation that cellularmechanisms may be the most efficient and effective way to transportdrugs and/or therapeutics across the blood brain barrier (BBB). While avariety of cellular transporter and receptor mechanisms have beenidentified, they are limited by the specificity of the ligand to theendothelial receptor and by both the affinity and avidity of the ligandfor the receptor. Further, identification of, as yet, unidentifiedantibody ligands and receptors will provide better tools by which totransport compounds across the BBB.

The inventors have mined a human single-chain antibody fragment (scFv)library for scFvs that bind to membranes of brain endothelial cells. Asubset of the scFv identified as binding to brain endothelial cells alsoendocytose into these cells, indicating that they may be substrates forBBB transcytosis systems. The scFv also specifically labels vessels ofall sizes in brain tissue sections in a pattern that represents atransport system of endothelial origin. No labeling is detected in otherbrain cells. This class of scFv and their associated BBB transportsystems represent a novel mechanism for noninvasive drug delivery to thebrain.

Before the present invention is described, it is understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the chemicals, cell lines, vectors, animals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

As used herein “subject” means mammals and non-mammals. “Mammals” meansany member of the class Mammalia including, but not limited to, humans,non-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, horses, sheep, goats, and swine;domestic animals such as rabbits, dogs, and cats; laboratory animalsincluding rodents, such as rats, mice, and guinea pigs; and the like.Examples of non-mammals include, but are not limited to, birds, and thelike. The term “subject” does not denote a particular age or sex.

As used herein, “administering” or “administration” includes any meansfor introducing compositions of the invention into the body, preferablyinto the systemic circulation. Examples include but are not limited tooral; buccal, sublingual, pulmonary, transdermal, transmucosal, as wellas subcutaneous, intraperitoneal, intravenous, and intramuscularinjection.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the BBB transcytosis compound (also termed“pharmaceutically active compound”) together with suitable diluents,preservatives, solubilizers, emulsifiers, and adjuvants, collectively“pharmaceutically-acceptable carriers.” As used herein, the terms“effective amount” and “therapeutically effective amount” refer to thequantity of active therapeutic agent or agents sufficient to yield adesired therapeutic response without undue adverse side effects such astoxicity, irritation, or allergic response. The specific “effectiveamount” will, obviously, vary with such factors as the particularcondition being treated, the physical condition of the subject, the typeof animal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compounds or its derivatives. In this case, anamount would be deemed therapeutically effective if it resulted in oneor more of the following: (a) the prevention of a neurological or braindisease (e.g., Alzheimers, Parkinson's and/or cancer); and (b) thereversal or stabilization of a neurological or brain disease (e.g.,Alzheimers, Parkinson's and/or cancer). The optimum effective amountscan be readily determined by one of ordinary skill in the art usingroutine experimentation.

Pharmaceutical compositions are liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the protein, complexation with metal ions, or incorporation ofthe material into or onto particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, milamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance. Controlled or sustained releasecompositions include formulation in lipophilic depots (e.g., fattyacids, waxes, oils).

Further, as used herein “pharmaceutically acceptable carriers” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.9% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia.

For purposes of the present invention, “treating” or “treatment”describes the management and care of a subject for the purpose ofcombating the disease, condition, or disorder. The terms embrace bothpreventive, i.e., prophylactic, and palliative treatment as well astherapeutic treatment. Treating includes the administration of acompound of present invention to prevent the onset of the symptoms orcomplications, alleviating the symptoms or complications, or eliminatingthe disease, condition, or disorder.

In one exemplary embodiment, the invention comprises an isolatedantibody fragment having the amino acid sequence set forth in any one ofSEQ ID NOs:1-34. In some preferred versions, the isolated antibodyfragment is a single chain fragment variable (scFv) fragment.

In another exemplary embodiment, the invention provides antibodies thatbind endothelial cell receptors resulting in endocytosis of the receptorand bound ligands. In some embodiments, the invention is apharmaceutical composition comprising an antibody linked to apharmaceutically active compound that is useful in transferring thepharmaceutically active compound across the blood brain barrier (BBB).

In another exemplary embodiment, the invention is an expression vectorthat includes a polynucleotide encoding the amino acid sequence setforth in any one of SEQ ID NOs:1-34. In other embodiments, the inventionincludes a purified and isolated host cell comprising the expressionvector containing the isolated nucleic acid encoding the amino acidsequence set forth in any one of SEQ ID NOs:1-34. It should beappreciated that the host cell can be any cell capable of expressingantibodies, such as, for example fungi, mammalian cells, including theChinese hamster ovary cells; insect cells, using, for example, abaculovirus expression system; plant cells, such as, for example, corn,rice, Arabidopsis, and the like. See, generally, Verma, R. et al., JImmunol Methods. 1998 Jul. 1; 216(1-2):165-81.

In yet another exemplary embodiment, the invention comprises a processfor expressing an antibody fragment capable of binding to a brainendothelial cell receptor comprising: (a) displaying an antibodyfragment on a yeast cell; (b) panning the antibody-displaying yeast cellagainst a brain endothelial cell culture; (c) identifying displayedantibody fragments that specifically bind brain endothelial cellreceptors; (d) inserting an isolated nucleic acid coding for theantibody fragment identified in (c) in an expression vector; and (e)transforming a host cell with the expression vector. In some versions ofthis embodiment, the host cell is selected from the group consisting of:yeast, bacteria, and combinations thereof. In some preferred embodimentsthe host cell is Saccharomyces cerevisiae or E. coli.

In another exemplary embodiment, the invention is a method ofidentifying a brain endothelial cell specific antibody. This embodimentincludes displaying antibody fragments on a yeast cell surface, panningthe displayed antibody against a brain endothelial cell culture,isolating specific binders to the membrane receptors on the brainendothelial cell, and identifying the specific binders, therebyidentifying an endothelial cell specific antibody. In some preferredembodiments the endothelial cell culture is provided in a cellmonolayer.

In still another exemplary embodiment, the invention comprises a methodof identifying endothelial cell receptors functioning in or related toendocytosis and transcytosis comprising, providing a culturedendothelial cell monolayer and panning a yeast-displayed antibodylibrary against the endothelial cell monolayer, isolating antibody boundendothelial cells; and identifying the cognate receptor.

Novel human antibodies that target BBB transport systems have immenseutility. Unlike the antibody delivery strategies that utilized wellstudied systems such as the transferrin and insulin systems, identifyingnovel receptor-mediated transport systems is quite difficult given thatthese ligands are membrane proteins that have yet to be identified.Therefore, the inventors have developed a novel selection methodology toidentify fully human antibodies having the required functionality of BBBbinding. The power of this technique is the concomitant selection ofpotential drug delivery vector and its cognate cell surface receptor(receptor-mediated carrier system) with no prior knowledge as to theidentity of either component. FIG. 4 depicts a general scheme for theselection of such antibodies according to the invention.

If antibodies are used to target receptor-mediated transport systems atthe BBB, then drug molecules and drug carriers can be effectivelytranscytosed across the BBB endothelium into brain tissue. Suchnoninvasive delivery from blood to brain is a result of the antibodyacting as a surrogate ligand for the endogenous transport systems.Current known antibody-targeted brain delivery systems include thetransferrin and insulin receptor systems. These receptors are expressedubiquitously throughout the body and lead to mistargeting of expensivepharmaceuticals.

Depending on the neuronal disorder targeted, a variety of brain drugcargoes, e.g. pharmacologic compounds or, equivalently, pharmaceuticallyactive compounds, can be delivered successfully in vivo usingantibody-based targeting according to the invention. As used herein, theterms “pharmaceutically active compound” and “pharmacologic compound”shall refer to any compound useful in treating or ameliorating theeffects of a disease or disorder. For example, diseases includingneurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, Huntington's disease, amyotrohpic lateral sclerosis (ALS, LouGehrig's disease) and multiple sclerosis can be targeted by use of suchdrugs as neurotrophic factors, including, but not limited to, nervegrowth factor (NGF), brain derived neurotrophic factor (BDNF), ciliaryneruotrophic factor (CNTF), glial cell-line neurotrohphic factor (GDNF)and insulin-like growth factor (IGF). In addition, other compounds thathave been shown to have therapeutic potential and may be delivered byantibodies of the invention are neuropeptides, including, but notlimited to, Substance P, neuropeptide Y, vasoactive intestinal peptide(VIP), gamma-amino-butyric acid (GABA), dopamine, cholecystokinin (CCK),endorphins, enkephalins and thyrotropin releasing hormone (TRH).Further, therapeutics may include cytokines, anxiolytic agents,anticonvulsants, polynucleotides and transgenes, including, for example,small-interfering RNAs which may be used for such neuronal disorders,including, but not limited to, psychiatric illnesses, such as, forexample anxiety, depression, schizophrenia, and sleep disorders, as wellas epilepsies, seizure disorders, stroke and cerebrovascular disorders,encephalitis and meningitis, memory and cognition disorders, paintherapeutics and physical trauma.

Antibodies are particularly well suited for targeting BBBreceptor-mediated transcytosis systems given their high affinity andspecificity for their ligands. As examples, appropriately-targetedantibodies that recognize extracellular epitopes of the insulin andtransferrin receptors can act as artificial transporter substrates thatare effectively transported across the BBB and deposited into the braininterstitium via the transendothelial route. Additionally, whenconjugated to drugs or drug carriers of various size and composition,the BBB targeting antibodies mediate brain uptake of the therapeuticcargo. Noninvasive transport of small molecules such as methotrexate hasbeen achieved using anti-transferrin receptor antibodies. Proteins suchas nerve growth factor, brain derived neurotrophic factor, and basicfibroblast growth factor were delivered to the brain after intravenousadministration by using an anti-transferrin receptor antibody. Thelatter two cases promoted reduction in stroke volume in rat middlecerebral artery occlusion models. Liposomes and liposomes containinggenes have also been delivered to the brain in vivo usinganti-transferrin receptor antibodies. In particular, gene-containingantibody-targeted liposomes have been targeted to rat brain forrestoration of tyrosine hydroxylase activity in an experimentalParkinson's disease model and to primate brain using a humanizedanti-insulin receptor antibody. In addition, brain delivery of the newclass of RNA interference drugs via pegylated immunoliposomes has beendemonstrated to increase survival of mice implanted with an experimentalhuman brain tumor model. In addition, anti-transferrin receptorconjugated nanoparticles have been produced. Finally, even if anantibody binds to the brain microvasculature or internalizes withoutfull transcytosis, it can have drug delivery benefits. When conjugatedto a liposome or nanoparticle loaded with lipophilic small moleculedrugs, it might be possible to raise the local BBB concentration of drugand help circumvent brain efflux systems, therefore facilitating brainuptake. Further, the ability to bind without internalization or withinternalization but without full trancytosis will provide for theidentification of BBB endothelial receptors or ligands that, once known,will allow for characterization and identification of the transportersystem and for optimization of antibodies that are internalized and/ortrancytosed. Taken together, these results indicate the utility ofantibody-targeted transcytosis systems for noninvasive trafficking ofdrugs into the brain.

As can be appreciated, one aspect of the present invention is in regardto antibody targets at the blood brain barrier, particularly, targetsthat represent the molecular machinery for internalization (endocytosis)and transcytosis. Accordingly, the present invention is directed to apurified antigen having a molecular weight of approximately 124 kDa whenimmunoprecipitated by human scFvA under non-reducing conditions. Theinventors identified and demonstrated this antigen is expressed inintact brain capillaries. The inventors have further demonstrated thatscFvB, scFvC, scFvG, and scFvK each recognize and are capable ofimmunoprecipitating the approximately 124 kDa antigen immunoprecipitatedby scFvA. Accordingly, scFvA, scFvB, scFvC, scFvG, and scFvK (SEQ IDNOs:1-5, respectively) are each useful as inventive blood braintargeting antibodies.

Accordingly, the invention further encompasses an antibody-blood brainbarrier transporter system. Such a system includes: (a) an antigenhaving a molecular weight of approximately 124 kDa whenimmunoprecipitated by human scFvA under non-reducing conditions, theantigen expressed in intact brain capillaries; and (b) a purifiedantibody bound to the antigen wherein said antibody has the amino acidsequence set forth in any one of SEQ ID NOs:1-5. The purified antibodyis preferably an scFvA antibody (SEQ ID NO:1), and, even morepreferably, the scFvA antibody is provided in combination with apharmaceutically active compound.

Furthermore, the inventors have identified neural cell adhesion molecule(NCAM) as an antigen that is immunoprecipitated by scFvJ antibody (SEQID NO:14). Accordingly, the invention further encompasses anantibody-blood brain barrier transporter system including: (a) anantigen comprising neural cell adhesion molecule (NCAM); and (b) apurified antibody bound to the antigen. In such a transporter system,the purified antibody binds to the NCAM antigen in the cell surface andis transported through the blood-brain barrier. In certain embodiments,the antibody included in the transporter system has the amino acidsequence set forth in SEQ ID NO:14. Preferably, the purified antibody isprovided in combination with a pharmaceutically active compound, and thetransporter system acts to facilitate the transfer of thepharmaceutically active compound across the blood-brain barrier.

Based upon the disclosed antibody-blood brain barrier transportersystem, the invention also contemplates a method of delivering apharmaceutically active compound across the blood brain barrier to asubject's brain. Such a method includes administering a pharmaceuticallyactive compound in combination with a purified antibody having an aminoacid sequence set forth in any one of SEQ ID NOs:1-5, SEQ ID NOs:11-12,SEQ ID NO:15, and SEQ ID NO:21 to a subject such that the antibodydirects delivery of the pharmaceutically active compound across theblood brain barrier to the subject's brain. The purified antibody ispreferably an scFv antibody having the amino acid sequence set forth inany one of SEQ ID NOs:1-5, SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:21,and the purified antibody is more preferably an scFvA antibody havingthe amino acid sequence set forth in SEQ ID NO:1.

In general, methods of conjugating, linking and coupling antibodies topharmacologically active compounds are well known in the field. Forexample, see, Wu A M, Senter P D, Arming antibodies: prospects andchallenges for immunoconjugates, Nat Biotechnol. 2005 September;23(9):1137-46 and Trail P A, King H D, Dubowchik G M, Monoclonalantibody drug immunoconjugates for targeted treatment of cancer, CancerImmunol Immunother. 2003 May; 52(5):328-37; Saito G, Swanson J A, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfidelinkages: role and site of cellular reducing activities, Adv Drug DelivRev. 2003 Feb. 10; 55(2):199-215. As well, the present antibodies may beprovided in combination with liposome, nanoparticles or other analogouscarriers loaded with a pharmaceutically active compound. Methods ofpreparing such compositions are known in the field (see, for example,Sugano et al., Antibody Targeting of Doxorubicin-loaded LiposomesSuppresses the Growth and Metastatic Spread of Established Human LungTumor Xenografts in Severe Combined Immunodeficient Mice Cancer Research60, 6942-6949, Dec. 15, 2000 and Martin et al., Nanomaterials inAnalytical Chemistry, Analytical Chemistry News & Features, May 1, 1998;pp. 322 A-327 A). As used herein, the phrase “antibody in combinationwith a pharmaceutically active compound” shall not be limited by themethod of manufacture and such compositions may be produced by, but notlimited to, techniques of conjugating, linking, coupling and decoratingknown in the art.

The examples described below disclose antibody delivery vectors that area critical component for disease-based applications. Identification ofsuch vectors combines an innovative combinatorial antibody screeningtechnology with expertise in the BBB and brain drug delivery fields toattack a complex delivery problem.

EXAMPLES

Various exemplary embodiments of compounds obtained as generallydescribed above and methods according to this invention will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the invention in any fashion.

Example 1 In Vitro Profiling of Brain Endothelial Cell Surface UsingLarge scFv Libraries

In order to simultaneously identify both brain endothelial cell surfaceproteins (potential BBB-resident transport systems) and antibodytargeting reagents (drug delivery vector), the inventors have performedvalidation of a novel cell surface profiling or “panning” technique.This technique uses a combinatorial library of naïve single-chainantibodies (scFv, FIG. 5) displayed on the surface of yeast to probe thesurface of endothelial cells. The antibody library contains ˜10⁹different scFv, and this diverse collection can be thought of as an “invitro immune system”. In this context, antibody-cell surface antigenpairs can be identified in a high throughput fashion. Yeast surfacedisplay has been well established as a technology for the directedevolution of antibody and T-cell receptors. The display system involvestethering single-chain antibodies to endogenous yeast mating agglutininsvia a polypeptide linker (FIG. 6). This platform results in the displayof 10⁴-10⁵ copies of a given scFv on the yeast cell surface where it isin prime position to sample the extracellular environment. Once a singleyeast clone is isolated from a library of antibodies, it acts as a“monoclonal” scFv producer. For the profiling of the endothelial cellsurface targeting efficacy, experiments were performed on model ligandsystems confirming that it is possible to perform yeast cell-endothelialcell screens. Similar screens were then performed to identify scFvsrecognizing endocytosing brain endothelial cell surface constituents.

Example 2 Yeast Cell-Endothelial Cell Targeting with a Model Ligand

The effectiveness of the panning method was first investigated using amodel scFv-surface ligand system (Wang, X. X. and Shusta, E. V. The useof scFv-displaying yeast in mammalian cell surface selections. J ImmunolMethods 2005, 304, 30-42) (incorporated herein in its entirety for allpurposes). Briefly, the hapten fluorescein was used as the surfaceligand and an anti-fluorescein scFv (4-4-20) was used as theyeast-displayed antibody. This system allowed detailed investigation ofthe factors governing panning success. Yeast displaying 4-4-20 exhibitedspecific interactions with fluoresceinated rat brain endothelial cells(RBE4 cell line Roux, F., et al., Regulation of gamma-glutamyltranspeptidase and alkaline phosphatase activities in immortalized ratbrain microvessel endothelial cells. J Cell Physiol 1994, 159, 101-13.)(FIG. 7) and could be recovered from large backgrounds of nonbindingirrelevant yeast (1 in 10⁶) in just three rounds. These high efficiencyselections required as few as 1700 fluorescein ligands per cellindicating the sensitivity and applicability to low abundance membraneproteins (transporters). Addition of free fluorescein competitor wasable to completely ablate the yeast-EC interactions, confirming thespecificity of the interaction.

The effects of scFv affinity on conjugate formation were examined sincethe naïve yeast library contains antibodies with affinities in thenanomolar range. The extent of enrichment was analyzed for dilutemixtures containing both 4-42-0 (Kd=1.3 nM) and a high affinity mutant4M5.3 (Kd=270 fM) [55] at varying cell surface FITC intensities.Briefly, 4-4-20:4M5.3:nonbinder mixtures of 1:1:10⁵ were applied toNAFITC-labeled monolayers having varying densities of fluoresceinligand. After a two hour incubation at 4° C., the wells were washed toremove non-specific binders. The simultaneous enrichment of 4-4-20 and4M5.3 was analyzed over three consecutive enrichment rounds (FIG. 7). At1,700 ligands per cell, both scFvs enriched with nearly equalefficiencies, while at the intermediate ligand densities, the 4M5.3enriched more rapidly. In these cases, the final product after threerounds contained between 15-25% 4-4-20 with the balance being the higheraffinity 4M5.3 scFv (FIG. 8). This result shows that it is possible toextract multiple scFv clones against a single cell surface target andalso suggests that scFvs against multiple antigens can be simultaneouslyenriched using this method. Therefore, the next step was to complete apanning selection against true endothelial antigens. An initial strategyfor cell culture panning of a yeast display library was developed asshown in FIG. 9.

Example 3 Overall Strategy for Identification of Blood BrainBarrier—Binding and Internalizing scFv

Recognizing the limitations of phage-display, the strategy illustratedin FIG. 9 was developed as a more powerful assay for the identificationof scFv's that bind brain endothelial cells. Briefly, as schematicallyrepresented in FIG. 9, the scFv library was mixed with an in vitroculture of brain endothelial cells. If a yeast cell has an antibodyagainst a surface protein on endothelial cells, the yeast cell can bindto endothelial cells under proper conditions. If the surface protein isat a higher level, the binding may be more robust. This allowed theyeast binders to be recovered to perform further binding andpurification. Specific binders were subjected to functionality tests todetermine if they can be endocytosed and trancytosed by endothelialcells and to further identify brain-epitope specificity. This is thefirst example of yeast-display antibody screening on whole cells. Thereare numerous advantages to performing antibody screening usingyeast-display (YSD) instead of phage-display. YSD has a very lowbackground binding, due to the flocculin-deficient strain of yeastexpressing the scFv library; in contrast, phage particles tend tononspecifically bind to mammalian target cells thereby givingfalse-positive binders. In addition the low background in YSD increasesthe signal to noise ratio thereby leading to fast enrichment of bindingclones and also the ability to identify clones that bind with lowaffinity. Yeast express from 50,000 to 100,000 scFv on their surfacewhile phage display antibody libraries express only 0-5 Ab/phage. Thislarge number of expressed surface scFv's allows for furtheridentification of weak binders due to the avidity effect which couldcause strong cumulative interactions between relatively low affinity Absand their antigens. Further, scFv displayed on yeast surface can beeasily produced as a soluble protein by shuttling the scFv expressioncassette into a secretion-expression vector. Thus, internalizationstudies of the scFv can be performed.

Example 4 Successful Screening of Human Nonimmune scFv Library for BrainEndothelial Cell Binding Clones

The inventors possess a human nonimmune scFv library in the identicalyeast display format as that used for the studies described inExample 1. Methods of making such a library are known in the art andhave been described by, e.g., Feldhaus et al. at Nat Biotechnol.21(2):163-70 (2003), incorporated by reference herein. The library iscomprised of ˜1×10⁹ scFvs that represent much of the in vivo humanantibody heavy and light chain gene diversity. Since the library isnaïve, it has not been subjected to any prescreening against antigen andthus can provide antibodies that bind a widely diverse range ofantigens. The library has been successfully used to isolate panels ofscFvs that bind with high affinity (K_(d)=1-1000 nM) to hapten antigens(fluorescein), peptide antigens having different phosphorylation states(p53), protein antigens (lysozyme), and the extracellular portion ofcell surface receptors (epidermal growth factor receptor). The yeast-ECtargeting method described in Example I was used for the profiling ofRBE4 rat brain endothelial cells using the scFv library. Briefly, theyeast library was applied to 100 cm² of RBE4 cells, and the bindingyeast clones recovered and amplified (FIG. 9). This process was repeatedfor three more rounds on 20, 10, and 10 cm² of RBE4 cells, respectively.The surface area of endothelial cells required decreases progressivelywith each round since the diversity of the library decreases. Thus,fewer yeast clones need to be examined to exhaustively cover theenriched pools. After just four rounds of selection, the number ofrecovered yeast was nearly equal to the number of yeast applied to theRBE4 cells indicating that almost the entire pool was comprised ofendothelial cell binding yeast clones (FIG. 10). Next, the individualclones from the recovered yeast pool were analyzed on a high throughputbasis to determine the identity of the scFvs mediating yeast-RBE4 cellbinding.

Example 5 Efficacy of Identification and Enrichment of RBE4-BindingscFv's via Biopanning

The inventors developed the biopanning antibody library screening methodbased on the model system described in EXAMPLE 2. This model was thenapplied to the YSD scFv library to identify BBB-binding andinternalizing antibodies. Preparation of the library is described byWang et al. at Nat. Methods, 4(2):143-5 (2007) which, along with thereferences cited therein, is incorporated by reference into the presentdisclosure. FIG. 10 illustrates the effects of multiple rounds ofenrichment using this method. As shown, after 4 rounds of panning, therewere a lot of yeast cells retained by the RBE4 cells compared to afterthe third round. Further, almost all of the input yeast population wasrecovered after the fifth round indicating that four rounds of screeningis apparently enough to “pull out” the BBB binders and thepresubtraction experiments against other cell types or endothelium couldbe used to screen for brain specificity. The recovery percentage of thelibrary is listed in the table in the lower panel of FIG. 10. Theefficacy of the method is illustrated by the increasing recoverypercentage of the library after progressive panning rounds. Incomparison, the values for the negative control 4-4-20 yeast stayed thesame.

Example 6 High Throughput Analysis of RBE4 Binding Competent YeastClones

While the capability of the inventors' panning system was illustrated bythe results shown in FIG. 10, the inventors still needed a method bywhich to rapidly review the clones in the library. Therefore, theinventors developed a method to allow the high-throughput screening andsequencing of binding clones. Briefly, yeast clones were grown in a96-well format such that scFv expression was induced on their surface(FIG. 11A). The yeast were then transferred directly to a 96-well platecontaining RBE4 cell monolayers (FIG. 11B). Subsequent to appropriatewashing, the 96-well plate was scanned by light microscopy. Yeast clonesthat truly mediate binding are simply identified by the retention ofyeast on the RBE4 cell monolayer (FIG. 11C). To ensure that endothelialcell binding is not simply a result of a spurious genetic mutation inthe yeast strain, a parallel plate was tested using yeast grown in acarbon source that does not allow induction of scFv expression (FIG.11D). After incubation and washing steps, the induced binder clones(FIG. 11C) had many yeast left but there were relatively few for thenoninduced yeast (FIG. 11D). The nonbinder clones had few yeast left forboth induced and noninduced groups. No yeast mutants were identified inthis particular screen and all binding was scFv mediated. Once the yeastbinders were identified, colony PCR was performed to amplify the openreading frame of each scFv clone (FIG. 11E). Briefly, the scFv-encodingplasmid was amplified directly from the yeast cells using restrictionenzyme BstNI. This enzyme cuts DNA frequently so different DNA wouldshow different digestion patterns or fingerprints (FIG. 11F). UniquescFv clones will generally exhibit a different restriction profile dueto the frequent occurrences of the five nucleotide BstNI recognitionsite. ScFvs having unique BstNI digestions were subject to sequencing.The PCR product of each clone exhibiting a unique restriction patternwas directly sequenced and the germline origin identified by IgGBLAST(FIG. 11G). A summary of unique clones is provided in the table shown inFIG. 12.

Example 7 Summary of Binding scFv Variants

FIG. 12 is a table summarizing binding of scFv variants. As shown, todate, a total of 2000 clones have been screened. Of the 2000 clones,1760 bound RBE4 cells. The binding clones are listed in FIG. 12. Theclones are organized such that the scFv's of the same germline usagesare clustered together. scFv's in each subset share high sequencehomology. However, regardless of the homology, CDRs varying in only 1amino acid may bind different antigens and thus, each may be uniquebinders. Further, even if the clones bound the same antigen they maybind with different affinity. In addition, it should be noted that someof the scFv's appear only once among all the clones screened. Thisillustrates the power of the high throughput screening method developedas such rare binders would otherwise not have been detected.

Example 8 Identification of scFv Germline Family

The germline family usages of these clones are summarized in FIG. 12.Unique scFv were clustered by homology. Each class of scFv antibodiesdiffers from all other classes by at minimum one CDR3 (<20% amino acidhomology). The CDR3 regions were focused upon for defining classes giventhe nonimmune basis for the library that limits CDR1 and CDR2 diversityand the fact that CDR3 of the VH and VL play a major role in determiningbinding specificity and affinity. Within a class, CDR1, CDR2 and CDR3(>85% amino acid homology) of VH and VL all have high homology. FIG. 12also shows that scFv A and D dominate in the analyzed clones. This wouldprevent the analysis of more clones so the inventors developed an insitu Northern blotting method to first identify scFv A and D and thenanalyze non-A or D clones using the high-throughput method.

A total of approximately 2000 clones from the fourth round of panninghave been analyzed using this method. Of the 34 unique scFvs identifiedthus far, only a few clones were recovered multiple times, and based onstatistical considerations, many unique clones remain in the enrichedround 4 pool. The germline origins of the heavy and light variableregions for the recovered clones are indicated in FIG. 12 and two clones(E and J) consist only of the heavy chain variable region. The completemethod allows for rapid analysis of 1000's of BBB binding scFv clones. Asequence alignment of the scFv A-like clones is given in FIG. 13, analignment of the scFv D-like clones is given in FIG. 14 and an alignmentof F with the A-like clones is given in FIG. 15.

Example 9 Binding and Internalization of scFv by RBE4

While the test for efficacy of the yeast library began with anidentification of the binding clones, their use in transcytosis furtherrequires that the scFvs could be internalized. Therefore, the scFv'swere also examined to see if they could be internalized by RBE4 cells.Because yeast are too big to be internalized the scFv was produced as asoluble protein in yeast cell culture. To do this, scFv gene in the YSDplasmid was subcloned into a secretion plasmid (described below inEXAMPLE 10) and the scFv was then secreted into the yeast cell culturemedium. Briefly, the scFv open reading frames for several of the scFvsin FIG. 12 were subcloned into a yeast expression system that yieldsmg/L levels of active, purified scFv (Shusta, E. V., et al., Directedevolution of a stable scaffold for T-cell receptor engineering. NatBiotechnol 2000, 18, 754-9; Shusta, E. V., et al., Increasing thesecretory capacity of Saccharomyces cerevisiae for production ofsingle-chain antibody fragments. Nat Biotechnol 1998, 16, 773-7; Shusta,E. V., et al., Yeast polypeptide fusion surface display levels predictthermal stability and soluble secretion efficiency. J Mol Biol 1999,292, 949-56). This system is regulated by the galactose-inducibleGAL1-10 promoter and includes c-terminal c-myc epitope and a sixhistidine epitope tag for purification.

FIG. 16 shows the results of running the soluble scFv's onpolyacrylamide gels (top panel), all exhibiting an appropriate size forscFv's ranging from 28-35 kDs with expression levels ranging from 1-4mg/ml. As shown in FIG. 17, when the RBE4 cells were labeled with thesesoluble scFvs, the A-group showed strong labeling of RBE4 cells andcould also be internalized. On the other hand, the D-group scFvs andscFvF showed binding to RBE4 cells but were not internalized. Theresults of these experiments are summarized in the bottom panel of FIG.16 and below.

Several of the class 1, 2, 3, 4, 5, 7, 10, 12, 15, 16, and 17 scFv wereevaluated for their ability to mediate cellular internalization. ThescFv were predimerized with the anti-cmyc epitope tag antibody toprovide the bivalency often required for receptor clustering andendocytosis. The dimerized scFv were then applied to living RBE4 cellsat 4° C. to yield cell surface labeling. Subsequently, the RBE4 cellswere shifted to 37° C. for 30 minutes to enable cellular trafficking(FIGS. 17 and 20, data not shown for classes 4, 5, 7, 10, 12, 15, 16,and 17). Class 1, 4, 7, and 12 scFvs were rapidly internalized intovesicular structures within the RBE4 cells, whereas class 2, class 3,class 5, class 10, class 15, class 16, and class 17 scFvs bound the RBE4surface but did not promote internalization. Control anti-fluoresceinantibody (4-4-20) exhibited no binding or uptake. As suggested by thedistinct internalization patterns, scFvA and OX26 do not compete for thesame transport system. In addition, the transporter-scFvA interaction isnot sensitive to glycosylation status as deglycosylation of RBE4 cells(mannosidase and neuraminidase) prior to binding and internalization hadno observable effect. Further, the high homology of the scFvA clones B,C, G and K, shown in FIG. 12, indicates that members of this family maybe equally effective in promoting internalization.

More specifically, RBE4 cells were labeled with pre-dimerized scFv A,scFv D, scFv 4-4-20 (dimerized to promote receptor clustering oftenrequired for endocytosis) or OX26 monoclonal antibody at 4° C. for cellsurface labeling and then shifted to 37° C. to promote cellulartrafficking The cells were then labeled with AlexaFluor 555 conjugatedanti-mouse IgG at 4° C. followed by AlexaFluor 488 conjugated anti-mouseIgG with or without cell permeabilization by saponin (SAP) treatment.Merged images of the AlexaFluor-labeled images are shown in FIG. 20.ScFvA exhibited both surface and intracellular labeling (compare scFvAlabeling with (top left) and without (top middle) saponin treatment). Incontrast, ScFvD (top right) exhibited only surface labeling upon saponintreatment. There was no labeling with 4-4-20 control scFv (bottomright). These results indicate that scFvA was rapidly internalized intovesicular structures within the RBE4 cells, whereas scFvD and scFvFbound the RBE4 surface but did not promote internalization. Similarresults to that seen for scFvA were observed for classes 4 (scFvH), 7(scFvE), and 12 (scFv4S21)). As a point of reference the OX26 MAb (FIG.20, bottom left) has been demonstrated to endocytose and transcytoseacross brain endothelial cells in vitro and in vivo. As noted, FIG. 17displays analogous data further illustrating micrographs of dualfluorescent staining of RBE4 binding scFv's showing specific binding andinternalization of scFv A by RBE4 cells but not scFv D.

The inventors determined that class 1 scFv exhibited a clear bindingsignal with either supernatant or purified material at 3-4 μg/mL, butclass 2 (scFvD) and 3 scFv (scFvF) required approximately 10-fold higherpurified concentrations of 20-80 μg/mL to yield cell surfaceimmunolabeling. In terms of binding affinity to live RBE4 cells, class 1scFvA possessed an affinity of Kd=82±15 nM, whereas the affinity ofclass 2 scFvD as a monomeric protein could not be determined using thismethod. Instead, the inventors evaluated the binding properties of scFvDafter predimerization with an epitope tag antibody (avidity=2.0±0.1 nM).As a comparison, the affinity of an anti-transferrin receptor scFvisolated using phagemid panning was measured to be 135 nM, of similaraffinity to scFvA isolated with our yeast panning system.

FIG. 18 provides equilibrium binding attributes of scFvA and scFvD. Theleft panel of FIG. 18 depicts the binding isotherm for scFvA interactionwith live RBE4 cells. The plot shows the fitted monomeric equilibriumbinding functions and experimental data from two independentexperiments. The right panel of FIG. 18 provides the binding isothermfor dimerized scFvD interaction with RBE4 cells. The plot shows thefitted monomeric equilibrium binding functions used to generate anapparent affinity (avidity) and experimental data from two independentexperiments. Insets illustrate raw flow cytometry histograms that wereused to generate the binding curves.

Thus, the results disclosed herein clearly indicate that yeast culturesupernatants can be used directly for facile biochemical testing similarto approaches that use hybridoma-conditioned medium as a source for MAb.Further, the scFv can be purified for labeling and transport studiesusing the c-terminal six histidine epitope as described in EXAMPLE 11.

Example 10 Immunoprecipitation of Antigens for scFv

To assess the nature of the antigens recognized by the scFv, theinventors developed a novel yeast immunoprecipitation procedure. Theyused yeast displaying scFv to directly immunoprecipitate the cognateplasma membrane antigens from detergent-solubilized, biotinylated RBE4lysates. Conveniently, the use of yeast as the immunoprecipitationparticle allowed the sizing of antigens without any additionalsubcloning, production or immobilization of scFv proteins required bytraditional immunoprecipitation methods. The inventors assessed theimmunoprecipitated products by anti-biotin Western blotting (FIG. 19),and the low amount of background in such blots was a direct indicator ofthe specificity of the immunoprecipitation process. The antigensimmunoprecipitated by class 1 scFvA (124 kDa-nonreduced, several largemolecular weight bands-reduced), class 2 scFvD (104 kDa-nonreduced, 117kDa-reduced) and class 6 scFvJ (122 kDa-nonreduced, 127 kDa-reduced)were distinct (FIG. 19). As predicted by the homology-based scFv classassignment, other class 1 scFv (scFvB, scFvC, scFvG and scFvK) yieldedimmunoprecipitation products identical to that seen for scFvA, and class2 scFv I like that observed for class 2 scFvD (data not shown). Themultiple bands appearing in the reduced scFvA sample suggest that alongwith the antigen recognized specifically by scFvA,co-immunoprecipitation of other biotinylated members of a proteincomplex or possibly multiple specific antigens may be occurring.Accordingly, scFv-antigen systems were identified that represent novelantibody-blood brain barrier transporter combinations that will form thebasis for drug delivery.

Example 11 Purified scFvA Labels the Brain Vasculature In Vivo

The high-throughput method described so far allowed the identificationof scFv clones that bind specifically to RBE4 cells and may beendocytosed in culture. However, the inventors wanted to confirm thatclones identified in culture also bound brain endothelial cells in vivo.Therefore, it was necessary to first confirm that the antigen targetedby scFvA in culture was also present on the endothelial cells in vivo.FIG. 21 depicts the assessment of scFvA target antigen density and brainlocalization. Specifically, the left panel illustrates flow cytometricassessment of antigen density on RBE4 cells. Anti-transferrin receptormonoclonal antibody (OX26), IgG2a isotype control (not shown),pre-dimerized scFvA, pre-dimerized scFvD, and pre-dimerized control4-4-20 scFv were used at antigen-saturating concentrations to label RBE4cells in order to make direct comparisons of antigen density. Therelative number of antigen sites present on the RBE4 cells was thenassessed by flow cytometry. The scFvA antigen was present atapproximately 3-fold higher levels than that found for scFvD, and5.5-fold greater than that observed for the transferrin receptor. Also,the OX26 monoclonal antibody did not compete with scFvA inimmunolabeling experiments, nor was the transporter-scFvA interactionsensitive to glycosylation status as deglycosylation of RBE4 cells(mannosidase and neuraminidase) prior to binding had no observableeffect.

The right panel of FIG. 21 demonstrates that ScFvA preferentiallyrecognizes the brain microvasculature. Co-localization of scFvA labelingwith the highly brain capillary-specific lectin, Griffonia simplicifoliaagglutinin (GSA) was complete and indicated that all brain blood vesselsexpress the scFvA antigen. Frozen rat brain sections were co-labeledwith scFvA (image 1) or 4-4-20 (image 4) and the brain endothelial cellmarker GSA-FITC (images 2, 5). Images 3 and 6 are merged imagesindicating the overlap between scFv and GSA-FITC labeling. In contrastto scFvA, irrelevant scFv 4-4-20 did not yield any labeling. Similarresults were observed for mouse brain sections and freshly isolatedcapillaries. Finally, much like the ranking of antigen density for RBE4cultures, qualitative labeling intensities indicated that the scFvAantigen density in vivo was higher than that for the transferrinreceptor (data not shown). The scale bar provided in image 3 is 50 μm inlength. Therefore, scFvA specifically labels the vascular component ofbrain tissue in vivo and the antigen is of endothelial origin.

Example 12 Materials and Methods

The following provides a detailed description of the materials andmethods utilized in the foregoing example sections.

Growth and Induction of scFv Library.

The nonimmune human scFv library in EBY100 yeast8 (a, GAL1-AGA1::URA3ura3-52 trp1 leu2Δl his3Δ200 pep4::HIS2 prblΔl.6R canl GAL) was grown at30° C. in SD-CAA (20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0g/L casamino acids, 10.19 g/L Na₂HPO₄.7H20, 8.56 g/L NaH₂PO₄ .H ₂O) plus50 μg/L kanamycin for 24 hours (OD600˜10). Yeast at 10-fold excess ofthe library diversity (5×10⁹) were subsequently induced in 500 mL SG-CAAmedium (same as SD-CAA except dextrose replaced by galactose) at 20° C.for 22 hours prior to panning against RBE4 monolayers.

Panning of scFv Library Against RBE4 Cell Monolayers.

The RBE4 rat brain endothelial cell line was used as the brainendothelial cell source as RBE4 cells have previously been demonstratedto display many attributes characteristic of the BBB in vivo. RBE4 cellsexhibit a nontransformed phenotype, express typical endothelial markers,respond to astrocyte cues, and exhibit BBB-specific properties such asthe expression and correct localization of the tight junction proteinoccludin. In addition, plasma membrane-localized transporterscharacteristic to brain endothelial cells including those that transportglucose (GLUT1), large neutral amino acids (LAT1), and iron (transferrinreceptor), and those that function in active efflux at the BBB(p-glycoprotein, MDR1), are expressed by RBE4 cells. RBE4 cells were akind gift from Dr. Françoise Roux and were maintained as describedpreviously. RBE4 cells were seeded on collagen type I-coated (Sigma)6-well plates at 25% confluency two days prior to panning Induced yeastcells at 10-fold excess of the library size (5×10⁹ yeast) were washedtwice with 0.01 M PBS, pH 7.4, supplemented with 1 mM CaCl₂, 0.5 mMMg₂SO₄ and 0.1% bovine serum albumin (BSA) (Wash buffer) and the yeastmixture was added dropwise onto 100 cm² of RBE4 cell monolayer to ensureeven distribution across the monolayer. The density of yeast (5×10⁷yeast/cm²) is at the upper limit for panning in that the yeastcompletely coat the RBE4 monolayer. Panning at high density allows ˜30%recovery of binding yeast while also providing appropriate oversamplingof the library diversity. The monolayers were then incubated at 4° C.for 2 hours to allow yeast-RBE4 cell contacting. The washing strategywas optimized to recover a model scFv that binds to RBE4 cells withnanomolar affinity. The resulting method involved washing the RBE4layers with ice cold wash buffer by gently rocking the plate twenty-fivetimes, rotating the plate five times (repeated twice), and rotating theplate ten times. The washing supernatant was removed after each step andreplaced with fresh wash buffer. After the washing steps, 1 mL of washbuffer was added into each well and all cells were scraped off the plateand pooled together. The yeast/RBE4 cell mixture was resuspended in 5 mLkanamycin-supplemented SD-CAA and grown at 30° C. overnight, followed bySG-CAA induction for 20 hours at 20° C. In parallel, a small fraction ofthe recovered cells were plated on SD-CAA agar plate to quantify thetotal number of recovered yeast cells after each round. Since the pooldiversity was greatly reduced after round 1, the yeast panning densitywas lowered to 5×10⁶ yeast/cm², and the RBE4 area was reduced to 20 cm²for round 2 and 10 cm² for rounds 3-5. After round 2 of panning, therecovered yeast clones numbered 8.2×10⁴, and a parallel experiment withcontrol yeast displaying an anti-fluorescein scFv (4-4-20) showed verylittle background using the same washing regimen indicating that thepanning strategy was yielding primarily RBE4-binding yeast clones. Toconfirm that the yeast-RBE4 interactions were scFv-based, thescFv-encoding plasmids for several RBE4-binding yeast clones (7 fromround 3, 12 from round 4) were recovered using the Zymoprep yeastminiprep kit (Zymo Research). The scFv-encoding plasmid was thenretransformed into yeast surface display parent strain, EBY100, usingthe lithium acetate method and Trp+ transformants were selected. AfterRBE4 binding with the retransformed clones was confirmed, the plasmidswere sequenced with the Gal1-10 (5′-CAACAAAAAATTGTTAATATACCT-3′; SEQ IDNO:35) and alpha terminator primers (5′-GTTACATCTACACTGTTGTTAT-3′; SEQID NO: 36) (UW-Madison Biotechnology Center).

High-Throughput Analysis of Recovered Yeast Clones.

As described above, yeast are typically grown first in SD-CAA followedby SG-CAA to promote scFv expression. However, this technique yieldedcomparatively low levels of scFv surface expression level and loweredpercentages of yeast displaying scFv when a 96-well format was used.Therefore, the scFv display methodology was optimized for 96-wellplates, and it was found that simultaneous growth and induction inSG-CAA allowed for scFv display having similar efficiency to thatobserved using the traditional yeast display methods. Thus, for highthroughput screening, yeast clones were inoculated into 200 μL of SG-CAA(induced sample) and SD-CAA (control uninduced sample) in a 96-wellplate and incubated at 30° C. for 24 hours. After removing 160 μL of SDculture to ensure similar total yeast numbers as the parallel SGculture, the 96-well plate of yeast was centrifuged, and the supernatantwas carefully removed. The yeast were then washed once with 150 μL washbuffer and resuspended in 150 μL wash buffer. In parallel, RBE4 cellscultured to confluency in a 96-well plate were washed once with ice-coldwash buffer. The yeast clones were then transferred into correspondingwells containing RBE4 monolayers and incubated at 4° C. for 2 hours.After washing, light microscopy was used to assess the binding capacityof the scFv yeast clones. After visual inspection, a yeast clone wasdefined as RBE4-binding if induced yeast remained bound while uninducedyeast originating from the same clone were washed away.

The scFv genes harbored by binding yeast clones were directly amplifiedby whole yeast cell PCR. Briefly, a small amount of a fresh, uninducedyeast colony was transferred into 30 μL 0.2% SDS, vortexed, frozen at−80° C. for 2 minutes and incubated at 95° C. for 2 minutes (temperatureshift repeated once). One microliter of the cell lysis solution was thenused as a PCR reaction template with primers, PNL6 Forward(5′-GTACGAGCTAAAAGTACAGTG-3′; SEQ ID NO: 37) and PNL6 Reverse(5′-TAGATACCCATACGACGTTC-3′; SEQ ID NO: 38). Subsequently, 20 μL of PCRproduct was subjected to BstNI (New England Biolabs) restriction digestat 60° C. for 14 hours. The digested products were resolved on a 3%agarose gel for unique scFv clone identification. The PCR product ofeach clone displaying a unique BstNI digestion pattern was sequencedwith Rev Seq P2 (5′-CCGCCGAGCTATTACAAGTC-3′; SEQ ID NO: 39) and For SeqP2 (5′-TCTGCAGGCTAGTGGTGGTG-3′; SEQ ID NO: 40) primers. The sequence wasthen analyzed by the IgBLAST program to identify the human germlineorigin (IgBLAST available at NCBI website: www.ncbi.nlm.nih.gov).

Yeast Colony Northern Blotting.

Yeast colony Northern blotting was used to detect and presubtract class1 and class 2 scFv from the yeast binding pool. Reagents and instrumentswere prepared as in standard Northern blotting experiments to eliminateRNase contamination. Yeast clones were cultured on SD-CAA agar platesand the resulting colonies were transferred onto ethanol-sterilizednitrocellulose membranes. The colony-loaded membrane was then layered ontop of SG-CAA agar plates, cell side facing up, and incubated at 30° C.for 2 days to induce transcription of the scFv gene. To prepare theinduced yeast colonies for Northern blotting, the nitrocellulosemembranes were layered onto Whatman filter paper soaked with 10% SDS andincubated at 65° C. for 30 minutes. The filters were then fixed bytransferring to formaldehyde-soaked filter paper at 65° C. for 30minutes (3×SSC, 10% formaldehyde in ddH₂O). Air-dried membranes weresubsequently baked for 2 hours at 80° C. under vacuum. Oligonucleotideprobes corresponding to Class 1 VHCDR2 and Class 2 VHCDR2 wereradiolabeled with a 10 residue 32^(P)-dATP tail using the STARFIRE kitaccording to manufacturer's instructions (IDT), and their specificradioactivity was determined by scintillation counting. Being part ofthe germline V-region, the VHCDR2 regions exhibited 100% homology withinclass 1 and class 2, and were therefore amenable to hybridization-basedsubtraction. The membranes were blocked in prehybridization buffer (50%formamide, 5× Denhardt's solution, 5× SSPE, 1% SDS, 0.1% salmon spermDNA) at 43° C. for 2 hours, and then hybridized (prehybridization bufferwith 8×105 cpm/ml of each probe) at 43° C. overnight. Afterhybridization, the nitrocellulose membranes were washed as follows:2×SSC, 0.1% SDS at room temperature for 8 minutes, 0.5×SSC, 0.1% SDS atroom temperature for 8 minutes, 0.1×SSC, 0.1% SDS at room temperaturefor 8 minutes, 0.1×SSC, 1% SDS at 50° C. for 30 minutes. The membraneswere then exposed to ECL Hyperfilm (Amersham) at −80° C. for 24 or 72hours. Although VHCDR2 was used as the probe in these subtractivescreens, the diversity of the recovered scFv clones can be readilyexpanded as desired via subtraction using any combination of CDR probes.

ScFv Secretion and Purification.

Open reading frames for scFv were isolated from the PCR products usedfor BstNI typing by NheI-HindIII restriction digest and were shuttled toan scFv yeast secretion vector (pRS316-GAL4-4-20) that has been usedextensively for scFv secretion. The resultant pRS316-GALscFv plasmidswere then transformed into YVH10, a yeast strain overexpressing proteindisulfide isomerase. Yeast harboring scFv secretion vector were grown inminimal SD medium (2% dextrose, 0.67% yeast nitrogen base) supplementedwith 2× SCAA amino acid (190 mg/L Arg, 108 mg/L Met, 52 mg/L Tyr, 290mg/L Ile, 440 mg/L Lys, 200 mg/L Phe, 1260 mg/L Glu, 400 mg/L Asp, 480mg/L Val, 220 mg/L Thr, 130 mg/L Gly, 20 mg/L tryptophan lacking leucineand uracil) at 30° C. for 72 hours. Subsequently, scFv secretion wasinduced at 20° C. for 72 hours in SG-SCAA (dextrose substituted bygalactose) with 1 mg/ml BSA as a nonspecific carrier. For experimentsrequiring purified scFv, Ni-NTA columns (Qiagen) were used to purify thesix histidine-tagged scFv from 50 mL or 1 L batches as describedpreviously in the literature.

The size, purity, and secretion yields of scFv were analyzed bySDS-polyacrylamide gel electrophoresis (PAGE) with a 4% stacking and12.5% separating gel followed by Coomassie blue staining Proteinconcentrations were estimated by comparison to a series of carbonicanhydrase standards (31 kDa) and by BCA protein assay (Pierce). Inparallel, the SDS-PAGE resolved proteins were also blotted onto anitrocellulose membrane (BioRad) for Western blotting. Thenitrocellulose membrane was blocked at 4° C. overnight in TBST solution(8 g/L NaCl, and 0.1% Tween-20, buffered to pH 7.6 with 20 mM Tris)supplemented with 5% nonfat milk and probed with 1 μg/mL 9E10 anti c-mycantibody (Covance) followed by an anti-mouse IgG horse radish peroxidaseconjugate (Sigma). Detection was performed using enhancedchemiluminescence and multiple time point exposures to ECL hyperfilm(Amersham) were evaluated by NIH ImageJ software for quantification.

Affinity Determination.

RBE4 cells were labeled at various scFvA concentrations at 4° C., andthe bound scFv detected by anti-cmyc (9E10) antibody labeling followedby anti-mouse IgG AlexaFluor 555. Fluorescence intensity was monitoredby FACSCalibur flow cytometer and used to quantitate fractional boundligand. The scFvA binding data was fit to an equilibrium binding modelto determine the monovalent affinity dissociation constant (Kd). Thecell-labeling assay was not sensitive enough to produce a binding curveusing monomeric scFvD, so scFvD was predimerized with anti-epitope tagantibody, 9E10, to provide the requisite avidity for the ligand bindingmeasurements. ScFv D dimer-labeled cells were then probed withanti-mouse IgG AlexaFluor 555 conjugate and assessed by flow cytometry.The resulting data were fit to an equilibrium binding model to derive anapparent affinity (avidity). For antigen density experiments, livingRBE4 cells were labeled at 4° C. with 62.5 nM of pre-dimerized scFvA,scFvD, 4-4-20 scFv, OX26 monoclonal antibody, or IgG2a isotype control.A uniform secondary antibody (anti-mouse IgG AlexaFluor 555 conjugate)was used for each sample to facilitate quantitative comparisons oflabeling intensity. These antibody labeling concentrations were adequatefor saturation binding of the cell surface antigens, and the cellsurface labeling was quantitatively assessed by flow cytometry.

ScFv-RBE4 Immunocytochemistry.

Predimerization of scFv via the c-myc epitope tag by the 9E10 antibodywas used as a method to provide bivalency which is often an importantcomponent for promoting cellular internalization of scFv. To this end,the RBE4-binding scFv was first incubated with 9E10 to form artificialdimers. Equal volumes of purified scFv (diluted to 8 μg/mL for scFvA or32 μg/mL for scFvD and 4-4-20 using 40% goat serum in PBS supplementedwith 1 mM CaCl₂, 0.5 mM Mg₂SO₄) and 10 μg/mL 9E10 were mixed andincubated at room temperature for 1 hour to form artificial dimer. RBE4cells at about 90% confluency were washed 3× with wash buffer. RBE4cells were then incubated with scFv artificial dimer or OX26 monoclonalantibody (10 μg/mL) (Serotec) at 4° C. for 30 minutes and then switchedto 37° for another 30 minutes. An anti-mouse IgG secondary antibodyconjugated with AlexaFlour555 (Molecular Probes) was applied for 30minutes at 4° C. to label cell surface-bound scFv. The cells were thenpermeabilized with 0.5% saponin (SigmaAldrich) diluted in wash buffer at4° C. for 5 minutes, and subsequently labeled with an anti-mouse IgGantibody conjugated with AlexaFluor488 (Molecular Probes) for 30 minutesat 4° C. to detect internalized scFv. Labeled cells were then fixed with4% paraformaldehyde and examined using a fluorescence microscope(Olympus IX70).

Yeast Display Immunoprecipitation.

ScFv-displaying yeast cells selected from the human scFv library weredirectly used to immunoprecipitate the cognate plasma membrane antigens.Yeast cells displaying anti-hen egg lysozyme (D1.3) scFv were used as anegative control. As a positive control, an anti-transferrin receptorOX26 scFv yeast display plasmid was created by excising OX26 scFv openreading frame from pRS316-GALOX26 as an NheI-XhoI fragment and ligatinginto pCT-LWHI. Yeast clones were grown and induced in 50 mL cultures asdescribed above. Induced yeast were collected by centrifugation, washedand fixed with 3% vol/vol formalin in PBS. RBE4 plasma membrane proteinswere biotinylated using 0.5 mg/mL Sulfo-NHS-LC-Biotin (Pierce). Toprepare RBE4 cell lysate, approximately 5×10⁶ biotinylated RBE4 cellswere lysed using a 1% (w/v) n-octyl-beta-D-glucopyranoside (scFvA, B, Cand J Sigma) or 0.1% (w/v) Triton X-100 (scFvD, I, J, and OX26, Sigma)detergent solution in PBS, supplemented with a protease inhibitorcocktail (Calbiochem). For immunoprecipitation, 400 μg of cell lysateprotein was mixed with approximately 10⁸ yeast cells and incubatedovernight at 4° C. Elution of immunoprecipitated product was performedby resuspending yeast cells in 30 μL of 0.5% SDS in 0.4 M Tris (pH 6.8)for 15 minutes. The eluates were separated with SDS-PAGE (8% separatinggel) with or without reducing agent (DTT) present, and blotted onto anitrocellulose membrane (BioRad). Western blotting was subsequentlyperformed with an anti-biotin monoclonal antibody (0.5 μg/mL, cloneBTN.4, Labvision), OX26 monoclonal antibody (5 μg/mL, Serotec), oranti-insulin receptor-subunit monoclonal antibody (1 μg/mL, clone CT-3,Labvision) as described above. Neither scFvA nor scFvD were active inWestern blotting format with immunoprecipitated products or with celllysates, likely a result of selections being performed under nativeconditions with living cells.

Immunohistochemical Labeling of Rat Brain Sections by scFv A.

Brain tissue sections were prepared from the brain of an adult maleSprague Dawley rat. The brain was snap-frozen with tissue freezingmedium (Triangle Biomedical Sciences) using a liquid nitrogen bath, and7 μm coronal sections were cut from the frozen brain. The brain sectionswere blocked with 40% goat serum and 0.2% TritonX-100 in PBSCM at roomtemperature for 30 minutes. Purified scFv A or 4-4-20 was diluted with40% goat serum and incubated with an equal volume of 10 μg/mL 9E10 for 1hour at room temperature to form artificial dimer. Brain sections werethen incubated with scFv A artificial dimer at 4° C. for 1 hour. Asecondary labeling solution consisting of phycoerythrin-conjugatedanti-mouse IgG and FITC conjugated Griffonia simplicifolia lectin(GSA-FITC 10 μg/ml, Sigma) was applied for 30 minutes at 4° C. Afterwashing, the brain sections were immediately fixed with 4%paraformaldehyde for 10 minutes on ice and examined by fluorescencemicroscopy.

Example 13 Improved Immunoprecipitation Method for Isolating andCharacterizing Antigens

In the above Examples, yeast library screening methods were used toidentify antibodies against cell surface antigens. Specifically, theinventors have demonstrated that yeast cells displaying scFvs can beused for immunoprecipitation and characterization by Western blotting oftarget cell (RBE04) surface antigens (see Examples 10 and 12). In thisExample, the inventors show the full capability of the yeast displayimmunoprecipitation technique (YDIP) for recovery and analysis of bothsoluble and plasma membrane antigens. Specifically, the inventors reportthe use of YDIP with tandem mass spectrometry to identify the RBE4plasma membrane antigen immunoprecipitated by scFvJ as the neural celladhesion molecule (NCAM).

Materials and Methods:

Cells, media and plasmids. Saccharomyces cerevisiae strain EBY100 wasused for surface display of scFvs. Surface display plasmids pCT201-D1.3and pCT302 were used for the display of anti-hen egg lysozyme D1.3 scFvand anti-fluorescein 4420 scFv, respectively. All human scFvs (scFvA,scFvD, scFvJ, and scFvK) in the surface display format were selectedfrom the previous examples. Yeast cells were grown in SD-CAA medium(20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids,10.19 g/L Na2HPO4.7H2O, 8.56 g/L NaH2PO4.H2O) at 30° C. to reach anOD600 nm of approximately 1.0 and induced in the same volume of SG-CAAmedium (dextrose replaced by galactose in SD-CAA) for 16-18 h at 20° C.for scFv display. The rat brain endothelial cell line (RBE4) was a kindgift from Dr. Françoise Roux. RBE4 cells were grown at 37° C. in 5% CO2,in 45% Alpha Minimum Essential Medium, 45% Ham's F10medium, and 10% heatinactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, Calif.)supplemented with 100 μg/mL streptomycin, 100 units/mL penicillin G(Invitrogen, Carlsbad, Calif.), 0.3 mg/mL geneticin (Fisher Scientific,Pittsburgh, Pa.) and 1 μg/L basic FibroblastGrowth Factor (bFGF) (RocheDiagnostics, Indianapolis, Ind.).

Preparation of biotinylated RBE4 cell lysates. Plasma membrane proteinsof RBE4 cells were biotinylated by incubating live RBE4 cells with 0.5mg/mL sulfo-NHS-LCBiotin (Pierce, Rockford, Ill.) in 10 mM phosphatebuffered saline (PBS, pH 7.4) supplemented with 1 mMCaCl2, 0.5 mM Mg2SO4(PBSCM) for 30 min with rocking at room temperature. The chargedsulfoxide group of the biotinylating reagent prevents the biotinylationof cytosolic proteins by hindering the diffusion through cell membranes.After the biotinylation, approximately 5×106 biotinylated RBE4 cellswere lysed at 4° C. by scraping the cells into 1 mL of PBS supplementedwith a protease inhibitor cocktail (Calbiochem, Gibbstown, N.J.), 2 mMEDTA and containing one of the following detergents: 0.1% (w/v) TritonX-100 (TX), 1% (w/v) n-octyl-β-D-glucopyranoside (OG) (Anatrace, Maumee,Ohio), 0.5% (w/v) CHAPS (Fisher Scientific), or radioimmunoprecipitationassay (RIPA) buffer (0.1% (w/v) SDS, 0.5% (w/v) sodiumdeoxycholate(DOC), and 1% (w/v) Triton X-100 in PBS). The initial cell lysates werethen centrifuged at 18,000×g for 15 min at 4° C. to remove insolubledebris. The total protein concentration remaining in the supernatant wasdetermined using the BCA assay per manufacturer's instructions (Pierce,Rockford, Ill.).

Detection of scFv activity in detergent solutions. To assess the effectsof detergents on scFv affinity, an anti-hen egg lysozyme antibody D1.3scFv and hen-egg lysozyme (HEL) from chicken egg white (Sigma, St.Louis, Mo.) were used. Yeast cells were fixed with 3% v/v formaldehydein PBS for 1 h at room temperature when indicated. The HEL wasbiotinylated using sulfo-NHS-LC-biotin (Pierce, Rockford, Ill.) and thedegree of biotinylation was determined to be 2.1 biotins per HEL usingthe 4-Hydroxyazobenzene-2-carboxylic acid (HABA) assay (Sigma, St.Louis, Mo.). Serial dilutions of HEL were prepared in the various lysatebuffers described above minus protease inhibitors and incubated withyeast cells displaying anti-HEL D1.3 scFv for 2 h at 20° C. Theincubated yeast cells were washed once with the corresponding detergentsolution, once with PBS containing 0.1% (w/v) bovine serum albumin(PBS-BSA) and subsequently incubated with a mouse anti-c-myc antibody(9E10, 30 μg/mL, Covance, Berkeley, Calif.) to selectively analyze theantibody-displaying yeast population. Next, goat antimouse IgG-Alexa488conjugate (αM488, 1/1000 dilution, Invitrogen, Carlsbad, Calif.) andstreptavidin-phycoerythrin conjugate (SAPE, 1/80 dilution, Sigma) wereadded to quantify ligand binding. All of the washing and labeling stepswere performed at 4° C. The fluorescence intensities were quantifiedusing the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes,N.J.) and fitted to a bimolecular equilibrium binding model to determinethe dissociation constants (Kd). To test the binding activity of scFvA,scFvK, scFvD, and scFvJ, yeast displaying scFv were incubated withbiotinylated RBE4 cell lysate (1% OG, 200 μg total protein) for 2 h at4° C. Yeast were then washed twice with PBS containing 1% OG. Labelingwith 9E10, SAPE, and αM488 was identical to that described above, exceptfor scFvJ, for which mouse anti-HA antibody (12CA5, Roche Diagnostics,Indianapolis, Ind.) was used instead of 9E10, since scFvJ is actually asingle domain Fv consisting of only the variable heavy chain and lackingthe C-terminal c-myc epitope. To measure the relative affinity of scFvAand scFvK, all procedures were identical as in measuring affinity ofD1.3 scFv, except that serially diluted RBE4 cell lysate (biotinylated,1% OG) was used as a ligand source.

Yeast Display Immunoprecipitation.

Immunoprecipitation from complex mixtures. Approximately 5×10⁷ of yeastcells displaying D1.3 scFvs were used for YDIP. Yeast cells were fixedwith 3% v/v formaldehyde in PBS for 1 h at room temperature whenindicated. Yeast were incubated 2 h at 4° C. with 34 nM HEL diluted intoRBE4 cell lysate prepared with 1% (w/v) TX and washed twice with andeluted with 50 μL of 0.2 M glycine-HCl solution (pH 2.0). Yeastdisplaying the anti-fluorescein 4-4-20 scFv were used as a negativecontrol.

Elution methods for antigen recovery. To monitor the amount of antigenobtained by various elution methods, D1.3 scFv and biotinylated HEL wereused. A batch of 3×10⁷ yeast cells displaying D1.3 scFv were incubatedwith 10 nM HEL in PBS-BSA for 2 h at 4° C. The yeast cells were washedtwice with PBS at 4° C. prior to elution. Elution of immunoprecipitatedproduct was performed by resuspending yeast cells in 30 μL of 0.2Mglycine-HCl solution (pH 2.0), 9 M urea, 0.2 M NaOH, 3 M NaCl, or 0.2%(w/v) SDS for 10 min at 4° C. Where indicated, yeast cells were fixedwith 3% v/v formaldehyde in PBS for 1 h at room temperature beforeincubation with the antigen, to reduce the co-elution of yeast proteins.The eluates were separated with SDS-PAGE (15% separating gel) and eitherprobed with streptavidin-HRP conjugate (1/2000 dilution, GE healthcare,Piscataway, N.J.) or silver-stained following standard protocols. TheHEL bands from Western blot films or silver stained gels were quantifiedusing densitometry with ImageJ (US National Institutes of Health,Bethesda, Md.) to compare the relative amount of biotinylated HEL elutedunder each condition.

Immunoprecipitaion of cell surface antigens and Western Blotting. ForWestern blotting analysis of immunoprecipitated antigens, 3-10×10⁷ yeastcells were used. Induced yeast cells were collected by centrifugationand washed with PBS-BSA. Biotinylated RBE4 cell lysate prepared usingthe various detergents containing approximately 400 μg of total protein(corresponding to approximately 2×106 RBE4 cells) was mixed with yeastcells and incubated overnight at 4° C. in a total volume of 200 μL.After the incubation, yeast cells were washed three times byresuspension in 1 mL of the corresponding lysis buffer (without proteaseinhibitor cocktail) and incubated for 15 min at 4° C. Elution ofimmunoprecipitated product was performed by resuspending yeast cells in30 μL of 0.2 M glycine-HCl solution (pH 2.0) for 10 min. The mixtureswere briefly centrifuged and the eluted supernatants were separated withnon-reducing SDSPAGE (8% separating gel), and blotted onto anitrocellulose membrane (BioRad, Hercules, Calif.). Yeast displayingD1.3 scFv were used as a negative control for these experiments. Westernblotting was subsequently performed with an anti-biotinmonoclonalantibody (0.5 μg/mL, clone BTN.4,Labvision, Fremont, Calif.). To testthe serial elution process, scFvJ was used. The IP procedure isidentical to that above except the eluted supernatant was seriallyapplied for the indicated number of times to elute additional batches ofyeast that have undergone the immunoprecipitation process. To confirmthe scFvJ antigen neural cell adhesion molecule (NCAM), a monoclonalanti-NCAM antibody (clone 5B8, Developmental studies Hybridoma Bank,Iowa Cities, Iowa) was used for Western blotting.

Identification of immunoprecipitated proteins using tandem massspectrometry. To sequence hen-egg lysozyme using tandem massspectrometry (MS/MS), approximately 3×108 yeast displaying the D1.3 scFvwere incubated for 2 h at 4° C. with 30 nM HEL in PBSTX, washed twicewith PBSTX and eluted with 50 μL of 0.2 M glycine-HCl solution (pH 2.0).The elution product was precipitated for 2 h by adding 250 μL ofice-cold acetone at −20° C. The protein pellet was dried andreconstituted in 5 μL of 8 M urea and then diluted with 40 μL of 50 mMammonium bicarbonate buffer with 10 mM DTT. Approximately 100 ng ofsequencing grade modified trypsin (Promega, Madison, Wis.) was added anddigestion was allowed to proceed for 16 h at 37° C. Peptide fractionswere individually analyzed by nanoLC-MS/MS using 1100 series LC/MSD TrapSL spectrometer (Agilent, Palo Alto, Calif.). Chromatography of peptidesprior to mass spectral analysis was accomplished using C18 reverse phaseHPLC trap column (Zorbax 300SB-C18, 5 μM, 5×0.3 mm, Agilent) andseparation column (Zorbax 300SB-C18, 3.5 μm, 0.075×150 mm, Agilent) ontowhich 40 μL of each extracted peptide fraction was automatically loaded.An Agilent 1100 series HPLC delivered solvents A: 0.1% (v/v) formic acidin water, and B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid ateither 10 μL/min, to load sample, or 0.28 μL/min, to elute peptidesdirectly into the nano-electrospray source over a 60 min 20% (v/v) B to80% (v/v) B gradient. As peptides eluted from theHPLC-column/electrospray source, MS/MS spectra were collected over 5channels from 300 to 2200 m/z; redundancy was limited by dynamicexclusion (120 s). MS/MS data were converted to mgf file format usingData Analysis Software (Agilent). Resulting mgf files were used tosearch the nonredundant NCBI database with an in-house licensed Mascotsearch engine (Matrix Science, London, UK) with methionine oxidation,cysteine carbamidomethylation and glutamine and asparagine deamidationas variable modifications.

For MS/MS sequencing of the RBE4 cell-surface antigen recognized byscFvJ, 3×109 formaldehyde fixed yeast cells displaying scFvJ weredivided into eight microfuge tubes and RBE4 cell lysate containing 3 mgof total protein (both biotinylated and non-biotinylated ID wassuccessful) in 1 mL of 1% OG with protease inhibitors was added to eachtube. The yeast cells were incubated overnight at 4° C. in the celllysate, and then 4 product pools created each by serial elution of twotubes with 70 μL of 0.2 M glycine-HCl solution (pH 2.0). To concentratethe antigen, the four elution products were combined and precipitated at4° C. for 2 h by the addition of trichloroacetic acid (FisherScientific) to a final concentration of 13% (w/v). Protein was pelletedby centrifuging at 12,000×g for 15 min at 4° C. The pelleted protein wasthen dissolved in 2× Laemmli sample buffer for separation by SDS-PAGE(8%). Gels were stained using colloidal coomassie to visualize theeluted proteins. In-gel digestion was performed using standardprotocols. In short, gel pieces were excised, dehydrated, and reduced in50 μL 25 mM DTT in 25 mM ammonium bicarbonate for 20 min at 56° C., andalkylated with 50 μL of 55 mM iodoacetamide (Sigma, St. Louis, Mo.) in100 mM ammoniumbicarbonate for 20 min at room temperature. Then the gelpieces were washed, dried and digested for 18 h at 37° C. with 400 ng ofsequencing grade modified trypsin in 25 mM ammonium bicarbonate with 5%(v/v) acetonitrile. Digested peptides were eluted off from a C18 microZipTip (Millipore, Billerica, Mass.) with acetonitrile/H20/TFA(60%:40%:0.2%) directly onto the Opti-TOF™ 384 Well plate (AppliedBiosystems, Foster City, Calif.) and re-crystallized with 0.75 μL ofmatrix Y. K. Cho et al./Journal of Immunological Methods 341 (2009)117-126 119 (10 mg/ml α-Cyano-4hydroxycinnamic acid inacetonitrile/H2O/TFA (70%:30%:0.2%)). Peptide identification via resultdependent MS/MS analysis was performed on a 4800 Matrix-Assisted LaserDesorption/Ionization-Time of Flight-Time of Flight (MALDI TOF-TOF) massspectrometer (Applied Biosystems, Foster City, Calif.). In short, apeptide fingerprint was generated by scanning the 700-4,000 Da massrange using 1500 shots acquired from 20 randomized regions of the samplespot using an OptiBeam™on-axis Nd:YAG laser (4200 intensity, 200 Hzfiring rate, 3-7 ns pulses). The fifteen most abundant precursors,excluding trypsin autolysis peptides and sodium/potassium adducts, wereselected for subsequent tandem MS analysis where 2000 total shots weretaken with 4700 laser intensity and 2 kV collision induced activation(CID) using air. Post-source decay (PSD) fragments from the precursorsof interest were isolated by timed-ion selection and reaccelerated intothe reflectron to generate the MS/MS spectrum. Raw data was deconvolutedusing GPS Explorer™ software and submitted for peptide mapping and MS/MSion search analysis against non-redundant NCBI database with an in-houselicensed Mascot search engine (Matrix Science, London, UK) with fixedmodification of carboxyamidomethylation of cysteines and parent/fragmention mass tolerance of 0.3 Da.

Quantitation of scFv and NCAM expression levels using flow cytometry. Toquantitate the expression level of scFvs on the yeast cell surface,Quantum simply cellular anti-mouse IgG microbeads (Banglabs, Fishers,Ind.), which contain four populations of beads with known mouse IgGbinding sites were used. Yeast cells displaying either D1.3 scFv orscFvA were incubated with a mouse anti-c-myc antibody (9E10, 30 μg/mL,Covance, Berkeley, Calif.) in PBS-BSA for 1 h. For scFvJ quantitation, amouse anti-HA antibody (12CA5, Roche Diagnostics, Indianapolis, Ind.,1/100 dilution) was used instead. The microbeads were also incubatedunder the same conditions. Next, the yeast cells and the microbeads werewashed, incubated with a goat anti-mouse IgG-Alexa555 conjugate (1/500dilution, Invitrogen, Carlsbad, Calif.) for 30 min. and analyzed usingthe FACSCalibur flow cytometer. To quantitate the expression level ofNCAM in RBE4 cells, RBE4 cells were permeabilized with 0.5% (w/v)saponin in PBSCM with 40% (v/v) goat serum (PBSCMG, Sigma, St. Louis,Mo.) and incubated with a mouse anti-NCAM antibody (clone 5B8, dilutedto 50 μg/mL) in PBSCMG for 1 h. The microbeads were also incubated underthe same conditions. The secondary reagents used were identical to thescFv quantitation.

Results:

Effect of Detergents on yeast-displayed scFv binding activity. Yeastdisplay immunoprecipitation (YDIP) takes advantage of scFv expressed asfusions to the yeast cell wall for capture of antigens from complexmixtures such as cell lysates (FIG. 22). Since creation of solubilizedprotein mixtures in the form of lysates requires the use of detergents(FIG. 22 step 1), particularly for the solubilization of membraneproteins, the scFv used in YDIP must retain their binding activities inthe presence of detergents. Thus, as a model system, the affinity ofyeast surface-displayed anti-hen egg white lysozyme (HEL) scFv (D1.3)for its HEL ligand was assessed in various detergent solutions.Detergents tested included 1% (w/v) Triton X-100 (TX), 1% (w/v)n-octyl-β-Dglucopyranoside (OG), 0.5% (w/v) CHAPS, and 0.1% (w/v) SDS aswell as a mixture of detergents (0.1% (w/v) SDS, 0.5% (w/v) sodiumdeoxycholate (DOC), and 1% (w/v) Triton X-100), which composes theradio-immunoprecipitation assay (RIPA) buffer. The concentrations ofdetergents were chosen to be higher than the critical micelleconcentration (CMC) of each detergent, since complete solubilization ofcell membranes generally occurs above the CMC. The D1.3 scFv remainedactive with comparable affinity for HEL to that found in PBS-BSA controlbuffer, varying by at most 3-fold (FIG. 23A, Table 1).

TABLE 1 Affinity of D1.3-HEL interaction in detergent solutions PBSadditives K_(d) (nM) BSA 1.07 ± 0.03 TX 0.488 ± 0.03  OG 2.61 ± 0.54CHAPS 1.80 ± 0.16 RIPA 3.43 ± 0.15 Reported Kd = 1.37 ± 0.14 nM(VanAntwerp and Wittrup, (2000)The lone exception was SDS, which resulted in a complete inhibition ofthe antigen-antibody interaction. Interestingly however, when 0.1% SDSwas mixed with 1% TX and 0.5% DOC as in the RIPA buffer, the scFvremained active (FIG. 23A). In addition to maintaining the nanomolarbinding affinity, it was important to determine the amount of antigenthat can be immunoprecipitated under each detergent condition, or inother words the percentage of scFvs that remain active. The levels ofantigen-antibody interaction were compared at saturating concentrationsof HEL (142 nM) in each detergent solution. Compared to the displayedscFvs binding to HEL in PBS-BSA, with the aforementioned exception ofSDS, more than 75% of the scFvs remained active in terms of the amountof antigen that can be immunoprecipitated (FIG. 23B).

ScFvs (scFvA, scFvK, scFvD, and scFvJ) that were previously selected ascapable of binding to rat brain endothelial cell line (RBE4) plasmamembrane proteins (see previous Examples) were used to assess thegeneral compatibility of yeast displayed scFv for immunoprecipitationboth from the standpoint of detergent tolerance and applicability tomembrane protein targets. Yeast cells displaying these scFvs were usedto immunoprecipitate antigens from RBE4 cell lysates generated by TXdetergent-solubilization of biotinylated plasma membranes (FIG. 22,steps 1-3). Using flow cytometry, significant antigen-antibodyinteraction was detected for yeast cells displaying scFvA, scFvK, scFvD,and scFvJ (FIG. 22, step 4) while no interaction was detected for yeastcells displaying an irrelevant scFv (FIG. 23C). Similar results wereobtained by using cell lysates generated by OG-mediated solubilization(data not shown and FIG. 23C inset). Moreover, Western blotting using ananti-biotin antibody indicates the presence of specificimmunoprecipitation products, and since biotinylated rat proteins arenot evident in the immunoprecipitation product using yeast displayingirrelevant antibody, the preparations are quite clean (FIG. 23C inset).In addition, since antigen-antibody interactions can be accuratelyquantified on the yeast surface, important antibody characteristics suchas relative affinity could be rapidly determined using YDIP (FIG. 22,step 4). As an example, scFvA and scFvK yield the sameimmunoprecipitation product (FIG. 23C inset) and these antibodies differby only six amino acids in the light-chain of CDR3 (FIG. 23D). Torapidly compare these two antibody clones, an affinity titration on thesurface of yeast using biotinylated cell lysates was performed and itwas determined that scFvA has a two-fold improved affinity compared withscFvK under these conditions (FIG. 23D). Notably, the relative affinitywas determined by direct titration of the cell lysate, therebyeliminating the process of antigen purification, which is often achallenge for cell-surface antigens. These results suggest that yeastdisplayed displaying scFvs retain their binding activities in variousdetergent solutions and that YDIP is effective in antigen isolation andcharacterization of antigen-antibody interactions.

Elution, antigen purity and quantity by YDIP. Next, we further assessedthe purity and quantity of antigens isolated using YDIP under a varietyof elution conditions. The anti-HEL D1.3 scFv was again tested as amodel system representing an antibody-soluble antigen combination. Wefirst sought to identify the most efficient method for dissociation ofbound antigens using YDIP of biotinylated HEL from a buffer solution(FIG. 22 step 5). Since it is known that various yeast cell wallproteins are also extracted from yeast under the elution conditionstested here, we assessed each elution condition not only in terms ofrecovered antigen quantity but also antigen purity relative tocoextracted yeast proteins. Tested elution conditions included low pH(0.2 M glycine-HCl, pH 2.0), high pH (0.2 M NaOH), 9 M urea, high salt(3M NaCl), and 0.2% (w/v) SDS. Each method resulted in elution ofantigen; however, it was immediately apparent that the amount ofcoextracted yeast protein was substantial (data not shown). Thus, toreduce the amount of coextracted yeast protein, yeast cells were firstfixed/crosslinked with a 3% formaldehyde solution prior to YDIP (FIG.22, step 2). Using fixed yeast cells for YDIP, there was no significanteffect on D1.3 scFv binding affinity and a small 10-20% decrease in HELcapture (FIGS. 23A and B). For elution of immunoprecipitated HEL fromfixed yeast, the low pH, SDS and urea elution methods had comparableefficiencies, while high pH and high salt were less effective (FIG.24A). In addition, the amount of coextracted yeast protein wassignificantly diminished by yeast fixation, with low pH elution havingthe least amount of coextracted protein (data not shown). Since low pHelution gave excellent yields and minimal yeast protein background, itwas generally used as the elution method.

In addition to coextracted yeast proteins, another confounding factorfor antigen analysis is the non-specific binding of mammalian celllysate proteins to yeast. Lysate-derived impurities can be moredetrimental than the coextracted yeast proteins in applications such asantigen identification, since yeast proteins can be distinguished frommammalian proteins by sequence in many cases, while contaminants fromthe same species cannot. To evaluate lysate-derived impurities, YDIP wasperformed to recover HEL that had been doped into RBE4 cell lysates.First, using unfixed yeast, several bands other than HEL were detectedon a silver-stained gel, especially in the lower molecular weight range(FIG. 24B lanes 1 and 2), yielding an HEL purity of approximately50-60%. For the most part, these proteins did not appear to belysate-derived as many of the same contaminants were detected in theYDIP of HEL in the experiments described above where lysate was absent(data not shown). Upon yeast fixation, the purity increased to 80% withboth yeast and lysate contaminants taken into account (FIG. 24B lane 4).Combined with the anti-biotin Western blotting results for scFvA, scFvK,scFvD, and scFvJ (FIG. 23C inset), these results show that proteins inRBE4 cell lysate have minimal non-specific binding to yeast. Finally,the immunoprecipitation of HEL from solution was specific to the yeastdisplaying D1.3 scFv and was not detected with yeast displaying anirrelevant scFv (FIG. 24B).

Another important aspect to consider is the amount of antigen isolated,because 50-500 ng are desired for characterization by peptide sequencingusing MS/MS. We first estimated the quantity of antigen that could bereasonably isolated using YDIP. Using the HEL-D1.3 pair that has anaffinity of Kd=0.488 nM in TX lysate (Table 1) with 31,000 scFv peryeast cell (see Materials and methods for scFv quantification details)and 10 nM or higher antigen concentration, approximately 150 ng ofantigen can be isolated with 3×108 D1.3-displaying yeast cells (15 mLovernight culture with density of 2 OD600 nm). Using 5×107 yeast cells(˜3 mL overnight culture), as was the case in FIG. 3B, it was thuspredicted that 25 ng of HEL could be immunoprecipitated from an RBE4cell lysate containing a 34 nM concentration of HEL. In practice,approximately 18 ng of HEL was immunoprecipitated (FIG. 24B lane 2)representing 70% of the theoretical yield. Therefore, using 10-100 mL ofovernight yeast display cultures for YDIP is sufficient to isolateantigens in amounts suitable for downstream characterization.

YDIP of mammalian cell-surface antigens. Since YDIP exhibited goodrecovery and purity for the soluble HEL antigen, the optimized YDIPprotocol was applied to analyze plasma membrane antigens. We have shownthat yeast cells displaying scFvs selected from a non-immune human scFvlibrary can be used to immunoprecipitate mammalian cell surface antigensfrom cell lysates for Western blot analysis using various detergents andelution conditions (FIG. 23). However, unlike the case of a solubleantigen like HEL, the plasma membrane antigen was being isolated fromcell lysates in low nanogram quantities that could not be detected byeither silver/coomassie staining or MS/MS. The reduced recovery ofantigen was likely a combined result of both antigen expression levelsand reduced antibody affinities (80-1000 nM) compared with the highaffinity D1.3 scFv. Therefore, to increase the YDIP yield as well asantigen concentration for downstream applications, we adapted a ‘serialelution’ method in which a single small volume of elution buffer is usedto serially elute multiple batches of yeast cells each used toimmunoprecipitate antigen from fresh cell lysate. Using scFvJ displayingyeast with OG-solubilized RBE4 cell lysate, YDIP was performed. Here, OGwas used for the lysis due to its compatibility with MS/MS analysis.ScFvJ immunoprecipitates an antigen of approximately 130 kDa (FIG. 23C),and serial elution effectively increased the antigen amount andconcentration nearly 20-fold over a single elution approach (FIGS. 25Aand B). After 4 elutions, little increase in eluted antigen is seensince the pH buffering capacity of 0.2M glycine-HCl becomes limiting.While antigen concentration increases dramatically, selectivity of theYDIP process is maintained as the level of irrelevant biotinylatedlysate proteins isolated during the procedure remains very low (FIG.25).

Identification of immunoprecipitated proteins using tandem massspectrometry. To assess the compatibility of YDIP with mass spectrometryfor peptide sequencing and antigen identification, we first tested theD1.3-HEL system. To this end, HEL was immunoprecipitated from a TXcontaining buffer using 3×10⁸ formaldehyde-fixed yeast cells. HEL wassubsequently eluted with low pH buffer, acetone precipitated and theentire elution product was trypsinized in solution. The trypsinizedelution product was directly subjected to LC-MS/MS to attempt detectionof HEL and to monitor the effects of yeast protein contaminants in theMS/MS analysis. As expected by the fact that HEL was isolated in highpurity (FIG. 24, lane 4) and in relatively large amounts, the HELantigen was unequivocally identified by the sequencing of four peptides.In addition, three other yeast proteins were identified including somescFv that were leached from the yeast surface during the YDIP procedure(Table 2).

TABLE 2 List of peptides sequenced by LC-MS/MS Protein HostUnique peptides sequenced Ion Score Hen egg white ChickenGTDVQAWIR (SEQ ID NO: 41) 33 GYSLGNWVCAAK (SEQ ID NO: 42) 43IVSNGNGMNAWVAWR (SEQ ID NO: 43) 104 NTDGSTDYGILQINSR (SEQ ID NO: 44) 83Single chain antibody Mouse AAAEQKLISEEDLN (SEQ ID NO: 45) 64ZPS1 (YOL154W) Yeast HYAGIDMLHR (SEQ ID NO: 46) 38LLNYGVDDVYYK (SEQ ID NO: 47) 23 KPLSTICFEGTIVDVGPK (SEQ ID NO: 48) 31QSAPAETVICDYFYTSK (SEQ ID NO: 49) 46CDDIDGLCAANPNYYAGHHR (SEQ ID NO: 50) 30 Glyceraldehyde-3- YeastIVSNASCTTNCLAPIAK (SEQ ID NO: 51) 56 dehydrogenaseTASGNIIPSSTGAAKAVGK (SEQ ID NO: 52) 40 ^(a) Proteins that have a summedpeptide ion score greater than 67 carry a significance of p < 0.05.

Next, YDIP was applied for de novo identification of the RBE4 plasmamembrane antigen immunoprecipitated by scFvJ. Although the molecularweight of the antigen was previously determined by anti-biotin Westernblotting (FIG. 23C), the identity of the antigen is unknown and thusprovides a true test of the full YDIP method. The in-solution digestionapproach used for HEL was not effective for this particular antigensince the amount of isolated antigen was smaller than that of HEL. Inaddition, 10-fold more yeast cells were required to isolate enoughantigen, which greatly increased the relative concentration ofcontaminating yeast proteins in the sample, thereby dominating the MS/MSsignal. Thus, after concentrating the immunoprecipitated antigens byserial elution followed by trichloroacetic acid precipitation andresuspension (see Materials and methods for details), the product wasfirst resolved by SDS-PAGE to separate the antigen from other proteinsin the elution mixture (FIG. 26A). The band at the size identified byWestern blotting and not present in a mock YDIP control sample, wasexcised and in-gel digested. Using tandem mass spectrometry (MS/MS), twopeptide sequences were obtained that identified rat neural cell adhesionmolecule (NCAM) as the immunoprecipitated product (FIG. 26B). The MS/MSsequencing result was further confirmed by Western blotting with ananti-NCAM antibody (FIG. 26C). The 130 kDa band corresponding to thebiotinylated immunoprecipitation product of scFvJ was also recognized byan anti-NCAM antibody. To get a feel for the antibody and antigendensities that allowed successful YDIP-coupled MS/MS identification,total cellular NCAM protein and the number of scFvJ molecules per yeastcell were quantified and found to be approximately 72,000 NCAM moleculesper cell (2 nM lysate concentration) and 47,000 scFvJ molecules peryeast, respectively (see Materials and methods for details).

Discussion:

This study demonstrates that scFv-displaying yeast cells are effectiveaffinity reagents allowing the immunoprecipitation and identification ofantigens from detergent solubilized cell lysates. We have shown thatyeast displaying scFvs retain their activities in detergent solutions byquantitative assessment of antigen-antibody interactions and two-folddifferences in antibody affinity could be accurately determined usingcell lysate as an antigen source. Furthermore, the immunoprecipitatedproteins could be readily characterized by well-established downstreamprocedures such as Western blotting, silver/coomassie staining, and massspectrometry. Optimized YDIP conditions that combine fixed yeast, a lowpH serial elution strategy and concentration by TCA precipitationallowed isolation of enough cell-surface receptor for MS/MS antigenidentification.

Although it is known that various non-ionic detergents such as TritonX-100, Tween 20, and Brij do not affect the activity of antibodies, thisgeneralization may not apply for antibody fragments such as scFvs thatcould be less stable than the whole immunoglobulin molecule. Here wehave shown that scFvs expressed on yeast surface are active in non-ionicdetergents (TX, OG), zwitterionic detergent (CHAPS), and a mixture ofionic and non-ionic detergents (RIPA buffer). It was important to test awide variety of detergents, since antigens have varying solubilitydepending on the detergent. Interestingly, while scFvs were inactive inthe highly denaturing ionic detergent, SDS, as previously seen forantibodies, when SDS was in a mixture with TX and DOC (RIPA buffer), thescFvs remained active. This might be explained by the fact that proteinsdenatured in SDS can be renatured by the addition of TX.

For the elution of antigens, we have applied conditions classically usedin immunoprecipitations (FIG. 24A). In the case of the HEL-D1.3 scFvpair, the low pH and Urea were most effective in dissociating theinteraction. However, the optimal elution may vary depending on thenature of antigen-antibody interaction. For example, it is thought thathydrophobic interactions are poorly disrupted by low pH conditions.Therefore, although the low pH elution conditions have been widely usedwith success, the elution method may need to be optimized for a givenantigen-antibody pair.

Another important consideration is the quantity of antigen isolated withYDIP. When the antigen concentration is in the nanomolar range, N200 ngof antigens could be easily isolated. However, as an example, thisantigen concentration corresponds to a protein expressed atapproximately 100,000 copies per mammalian cell for the case of a 14 kDaprotein, with 5×107 mammalian cells in 1 mL of lysis buffer. Thisconcentration of antigen may not be achievable in every situation, sincenormal protein expression levels may vary between 100 and 100,000 copiesper cell, averaging about 50,000 copies per cell, with plasma membraneproteins being on the low end of this spectrum. In the case ofscFvJ-NCAM pair that was evaluated in this study, quantification of NCAMexpression level in RBE4 cells showed that there are 72,000 copies ofNCAM per RBE4 cell corresponding to a 2 nM antigen concentration in thecell lysate. This result suggests that for antigens with averageexpression level, YDIP can be applied for de novo identification. On theyeast side of the equation, when the antibodies are isolated from a nonimmune or synthetic library, Kd values can be in the low micromolarrange limiting recovery from dilute cell lysates. Theoreticalcalculations based on bimolecular equilibrium show that even withantibodies with micromolar affinities, it is estimated that N100 ng ofantigens can be isolated using 109 yeast cells. Indeed this was the casefor the scFvJ-NCAM system where 3×109 yeast cells from 150 mL ofovernight culture were sufficient. In terms of scFv expression levelswhich can also affect YDIP yield, quantification of displayed D1.3,scFvA and scFvJ showed expression levels ranging from 31,000 to 68,000scFv per yeast cell, differences that can be accommodated for simply byscaling the number of yeast used in the YDIP procedure.

The major immunoprecipitation product that appears in anti-biotinWestern blotting of the scFvJ elution product was identified to be theneural cell adhesion molecule (NCAM) (FIG. 26). NCAM is a transmembraneprotein with immunoglobulin-like domains that has three alternativelyspliced isoforms of varying predicted molecular weights (120, 140, and180 kDa). Since the antibody that was used to confirm the YDIP result isspecific to the cytosolic domain of NCAM, which is absent in the 120 kDaisoform, it is likely that scFvJ immunoprecipitates the 140 kDa isoform.NCAM is known to be involved in cell-cell adhesion and neuriteoutgrowth. While NCAM is mainly found in early embryonic cells, neuralcells, and natural killer cells, it has previously been detected in geneprofiling studies as being expressed at the blood-brain barrier. Ourfinding that the actual protein is expressed in a rat brain endothelialcell line (RBE4) model of the blood-brain barrier confirms the geneprofiling study and suggests an endothelial role for NCAM in brainfunction. Since scFvJ was raised against intact cells, a cell surfacebiotinylation protocol was used, there was a single biotinylatedimmunoprecipitation product and the sequenced product is a membraneprotein with an extracellular epitope, NCAM is most probably the actualantigen recognized by scFvJ. However, we cannot rule out the possibilitythat scFvJ interacts with another member of an immunoprecipitatedprotein complex of which NCAM is a biotinylated member. ScFvJ wasselected under native, live cell conditions and does not recognize itsantigen under the denaturing conditions of Western blotting, and thus wecould not show a direct interaction between scFvJ and NCAM. This levelof confirmation is dictated by the scFv's properties and is thereforenot a challenge unique to YDIP. Nonetheless, our results suggest thatYDIP can be generally applied for antigen/antigen-complex identificationfor antibodies originating from cell surface screens.

In conclusion, the results show that YDIP provides an efficient andfacile method for antigen identification compared to conventionalimmunoprecipitation approaches since it eliminates additional cloningand production steps needed to obtain affinity reagents. Therefore, weanticipate that YDIP will enable a wide range of applications incombination with yeast antibody library screening technology.

As described in the preceding EXAMPLES, the invention provides a methodof panning a yeast display antibody library against cultured endothelialcells and simultaneously isolating multiple binders of differentaffinities against one or more antigens. The binding clones wereisolated and their ability to trigger endocytosis and transcytosis wasthen confirmed using endothelial cells. The invention also provides animproved immunoprecipitation method capable of identifying specificantigen targets. Using the improved immunoprecipitation method, theantigen binding target for scFvJ was identified to be the neural celladhesion molecule (NCAM).

While this invention has been described in conjunction with the variousexemplary embodiments outlined above, various alternatives,modifications, variations, improvements, and/or substantial equivalents,whether known or that are or may be presently unforeseen, may becomeapparent to those having at least ordinary skill in the art.Accordingly, the exemplary embodiments according to this invention, asset forth above, are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinvention. Therefore, the invention is intended to embrace all known orlater-developed alternatives, modifications, variations, improvements,and/or substantial equivalents of these exemplary embodiments.

Sequence Listing. Applicants are submitting as part of this Applicationa computer readable sequence listing txt file, which is incorporated byreference herein.

1. A method of delivering a pharmaceutically active compound to asubject's brain, comprising administering to a subject apharmaceutically active compound in combination with an antibody orantibody fragment that is specific to neural cell adhesion molecule(NCAM), whereby the NCAM directs delivery of the pharmaceutically activecompound across the blood brain barrier to the subject's brain.
 2. Themethod according to claim 1, wherein the antibody is a single chainfragment variable (scFv) antibody.
 3. The method according to claim 1,wherein the antibody or antibody fragment comprises the amino acidsequence set forth in SEQ ID NO:14.
 4. A composition comprising anisolated antibody or antibody fragment that is specific to neural celladhesion molecule (NCAM) in combination with a pharmaceutically activecompound.
 5. The composition according to claim 4, wherein the antibodyis a single chain fragment variable (scFv) antibody.
 6. The compositionaccording to claim 4, wherein the antibody or antibody fragmentcomprises the amino acid sequence set forth in SEQ ID NO:14.
 7. Anisolated polypeptide comprising an antibody fragment comprising theamino acid sequence set forth in SEQ ID NO:14.